Novel Strategies for Improved Cancer Vaccines

ABSTRACT

The present invention concerns methods and compositions for forming anti-cancer vaccine complexes. In preferred embodiments, the anti-cancer vaccine complex comprises an antibody moiety that binds to dendritic cells, such as an anti-CD74 antibody or antigen-binding fragment thereof, attached to an AD (anchoring domain) moiety and a xenoantigen, such as CD20, attached to a DDD (dimerization and docking domain) moiety, wherein two copies of the DDD moiety form a dimer that binds to the AD moiety, resulting in the formation of the vaccine complex. The anti-cancer vaccine complex is capable of inducing an immune response against xenoantigen expressing cancer cells, such as CD138 neg CD20 +  MM stem cells, and inducing apoptosis of and inhibiting the growth of or eliminating the cancer cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/754,740, filed Apr. 6, 2010, which is a continuation-in-part of U.S.patent application Ser. No. 12/544,476 (now U.S. Pat. No. 7,901,680),filed Aug. 20, 2009, which claimed the benefit under 35 U.S.C. 119(e) ofProvisional U.S. Patent Application Ser. No. 61/090,487, filed Aug. 20,2008, and which was a continuation-in-part of U.S. patent applicationSer. No. 12/396,605 (now U.S. Pat. No. 7,858,070), filed Mar. 3, 2009,which was a divisional of U.S. Pat. No. 7,527,787, which was acontinuation-in-part of U.S. Pat. Nos. 7,550,143; 7,521,056; and7,534,866; which claimed the benefit under 35 U.S.C. 119(e) ofprovisional U.S. Patent Application Nos. 60/782,332, filed Mar. 14,2006; 60/728,292, filed Oct. 19, 2005; 60/751,196, filed Dec. 16, 2005;and No. 60/864,530, filed Nov. 6, 2006. This application claims thebenefit under 35 U.S.C. 119(e) of Provisional U.S. Patent ApplicationSer. No. 61/168,290, filed Apr. 10, 2009, incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Apr. 5, 2010, is namedIMM319US.txt, and is 17,283 bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions and methods for improvedvaccines. In particular embodiments, the compositions and methods relateto xenoantigens, chemically modified antigens, antigens in conjunctionwith Th epitopes, dendritic cells loaded with antigens, immunoconjugatesor fusion proteins comprising dendritic cell targeting antibodiesattached to antigens and/or dendritic cell targeting lentiviral vectorsexpressing antigens. The vaccines are of use for therapy of a widevariety of diseases, including but not limited to cancer, autoimmunedisease, immune dysfunction disease, metabolic disease, neurologicaldiseases such as Alzheimer's and cardiovascular disease.

2. Related Art

Therapeutic vaccination against cancer is an important modalitycomplementing current standard therapies, and may lead to long-termcontrol of cancer. Numerous strategies are in development in an attemptto achieve better effectiveness, but except for the recent advent ofvaccines against HPV, the long effort to produce a cancer vaccine hasnot succeeded.

Two major categories of tumor-related proteins can be exploited aspotential antigens for cancer vaccines: tumor-specific antigens (TSAs)(Srivastava and Old, Immunol Today 9:78-83, 1988; Melief et al., ColdSpring Harbor Symp Quant. Biol 65:597-803, 1989; Hislop et al., Annu RevImmunol. 25:587-617, 22007) and tumor-associated antigens (TAAs)(Dalgleish and Pandha, Adv Cancer Res 96:175-90, 2007; Finn, NEJM358:2704-15, 2008). TSAs are molecules unique to cancer cells, such asthe products of mutated normal cellular genes, viral antigens expressedon tumor cells, endogenous human retrovirus activated and expressed incancer cells (Takahashi et al., J Clin Invest 118:1099-1109, 2008), anda recently identified placenta-specific antigen that is not found in anyadult normal somatic tissue but highly expressed in a variety of tumortypes, particularly in breast cancer (Chen et al., Beijing Da Xue XueBao 38:124-27, 2006; Koslowski et al., Cancer Res 67:9528-34, 2007; Old,Cancer Immunity 7:19, 2007). TAAs are molecules shared, but differentlyexpressed, by cancer cells and normal cells.

Although TSAs are the ideal targets for immunotherapy and vaccination,the fact that they are expressed only on individual patients' cancercells or small subsets of tumors would require the development ofpersonalized therapy for individual patients, thus limiting their wideapplication (Baxevanis et al., Cancer Immunol Immunother 58:317024,2009). As for TAAs, which are largely self-antigens (self-Ags) alreadytolerized by the immune system through a tightly controlled process ofnegative selection in the thymus (Kruisbeek and Amsen, Curr Opin Immunol8:233-44, 1996; Stockinger, Adv Immunol 71:229-65, 1999) or peripheraltolerization, the major challenge is to induce a strong immune responsedirected selectively against cancer cells.

A need exists in the field for new approaches to vaccines of use forcancer treatment and/or prevention, capable of inducing a strong immuneresponse against cancer cells and of wide application.

SUMMARY OF THE INVENTION

The present invention concerns novel approaches to the development ofeffective cancer vaccines, including but not limited to optimizedantigen design, in vivo targeted dendritic cell vaccination, and cancervaccines used in combination with chemotherapy, monoclonal antibodies,adoptive T-cell transfer, or stem cell transplantation. The rationaleand development of each of these approaches for improved cancer vaccinesis described below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Diagram of several novel strategies/approaches in cancervaccination. To overcome immune tolerance, antigens to be used forcancer vaccines are optimized which includes: (1) using xenoantigenother than TAA/self antigen, (2) chemically-modified TAA, (3) inclusionof antigen-specific CD4 Th epitopes. The optimized antigen is loaded todendritic cells followed by optimized maturation (αDC1) for ex vivo DCvaccination. The antigen can also be linked to a DC-targeting antibodyvia genetic fusion or chemical conjugation. In vivo administration ofthe conjugate or fusion protein specifically delivers the antigen to DCsand initiates immune responses. The optimized antigen can also beengineered with a lentivector which is pseudotyped with an envelopeprotein that can specifically recognize DCs (for example, a DC-SIGNantibody). In vivo administration of this recombinant lentiviral vectorselectively infects DCs. The antigen can also be encoded by a lentiviralvector which carries a lineage-specific internal promoter (e.g.,dectin-2 gene promoter) that restricts the transgene (antigen)expression to antigen-presenting cells. All of the vaccinationapproaches initiate antigen-specific (both CD4+ and CD8+) T cellresponses against cancer cells and/or cancer stem cells. Thetumor-specific CD4+ T cells provides help and survival signals totumor-specific CD8+ T cells and maintain CD8+ T-cell memory, which areessential for long-lasting anti-tumor immunity. The tumor protectionefficacy can be augmented by combinational use of adoptive transfer ofantigen-specific T cells, chemotherapy, monoclonal antibodies, and/orstem cell transplantation. Arrows with X represent inhibitory action, ornegative effect.

FIG. 2. Specific binding of hLL1 on human blood DC subsets, B cells, andmonocytes. (A) The gating strategy for the different APC subsets. (B)CD74 expression in APCs. (C) The binding efficiency of hLL1 on thecells. The numbers represent mean fluorescence intensity.

FIG. 3. CD74 expression in and binding efficiency of hLL1 with humanmonocyte-derived immature vs mature DCs. The human monocyte-derived DCs(day 5 after culture in the presence of hGM-CSF and hIL-4) were stainedwith FITC-labeled anti-CD74 antibody or AlexaFluor488-labeled hLL1, incombination with the staining with fluorescence-labeled mAbs againstHLA-DR and CD83. The HLA-DR-positive cells are gated and analyzed. (A)CD74 expression in immature and LPS-matured DCs. (B) hLL1 binding withimmature vs LPS-matured DCs. (C) Comparison of expression of CD83,HLA-DR, CD74 and hLL1 binding in immature and mature DCs.

FIG. 4. Side-by-side comparison of the cytotoxic effect of hLL1 on Bcell malignant Daudi cells and normal DCs. (A) Comparison of the effectof hLL1 on Daudi and DCs. (B) Effect of hLL1 on cell viability of DCs inan extended doses. (C) The cytotoxic effect of hLL1 on Daudi cells. (D)The microscopic image shows no effect of hLL1 on DC viability.

FIG. 5. Moderate enhancement of DC constitutive maturation by hLL1. TheHLA-DR positive cell populations were gated from day 5 DCs derived fromhuman monocytes in the presence of hGM-CSF and hIL-4. (A) The expressionof antigen-presenting molecule HLA-DR, costimulatory molecule CD54 andCD86 was measured by flow cytometry. (B) Expression levels ofantigen-presenting molecule HLA-DR, costimulatory molecule CD54 andCD86.

FIG. 6. No significant influence of hLL1 on DC-mediated T cellproliferation. The hLL1-treated DCs were co-cultured with CFSE-labeledallogeneic PBMCs for 8 (A) or 11 days (B). The expanded T cells werestained with Percp-conjugated mAb against CD4. The cell proliferation oftotal T cells, CD4+ and CD4− T cells were analyzed.

FIG. 7. Polarization of naïve CD4+ T cells by hLL1-treated DCs favoringthe differentiation toward Th1 effector cells. Naïve CD4+ T cellsisolated from human PBMCs using the depletion column with magnetic beads(MACS) were co-cultured with hLL1-treated allogeneic DCs. Afterdifferent time points (day 11, 13, 18), the cells were harvested,stimulated with PMA and ionomycin, and analyzed with intracellularcytokine staining with fluorescence-labeled hIFN-gamma and hIL-4antibodies. Th1/Th2/Th0 cells populations were gated and analyzed. Theflow cytokine production in T cells stimulated by hLL1-treated DCs or byGAH-cross-linked hLL1-treated DCs was determined. (A) The data of Th1responses in two donors, in the absence or presence of cross-linking byGAH, at different days after DC/T coculture, are shown. (B) Thedose-effect curve for increasing Th1 populations by hLL1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “a”, “an” and “the” may refer to either thesingular or plural, unless the context otherwise makes clear that onlythe singular is meant.

As used herein, the term “about” means plus or minus ten percent (10%)of a value. For example, “about 100” would refer to any number between90 and 110.

An antibody refers to a full-length (i.e., naturally occurring or formedby normal immunoglobulin gene fragment recombinatorial processes)immunoglobulin molecule (e.g., an IgG antibody) or an immunologicallyactive, antigen-binding portion of an immunoglobulin molecule, like anantibody fragment. In various embodiments, antibodies may be murine,chimeric, humanized or human, polyclonal or monoclonal, monospecific,bispecific or multispecific.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-HLA-DR antibody fragment binds toHLA-DR The term “antibody fragment” also includes isolated fragmentsconsisting of the variable regions, such as the “Fv” fragmentsconsisting of the variable regions of the heavy and light chains andrecombinant single chain polypeptide molecules in which light and heavyvariable regions are connected by a peptide linker (“scFv proteins”). Asused herein, the term “antibody fragment” does not include portions ofantibodies without antigen binding activity, such as Fc fragments orsingle amino acid residues.

A naked antibody or naked antibody fragment refers to an antibody orantigen binding fragment thereof which is not conjugated to atherapeutic agent. Naked antibodies may include murine monoclonalantibodies, as well as recombinant antibodies, such as chimeric,humanized or human antibodies.

A therapeutic agent is a molecule, atom or complex which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,and is useful in the treatment of a disease. Non-limiting examples oftherapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents, oligonucleotides (e.g. anti-senseoligonucleotides or siRNA) and radioisotopes.

An immunoconjugate is a conjugate of an antibody component with at leastone therapeutic or diagnostic agent. An antibody component may beconjugated with multiple therapeutic and/or diagnostic agents to form animmunoconjugate.

The term antibody fusion protein may refer to a recombinantly producedantigen-binding molecule in which one or more of the same or differentsingle-chain antibody or antibody fragment segments with the same ordifferent specificities are linked. Valency of the fusion proteinindicates how many binding arms or sites the fusion protein has to asingle antigen or epitope; i.e., monovalent, bivalent, trivalent ormultivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindwith one epitope. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators and toxins.One preferred toxin comprises a ribonuclease (RNase), preferably arecombinant RNase. However, the term is not limiting and a variety ofprotein or peptide effectors may be incorporated into a fusion protein.In another non-limiting example, a fusion protein may comprise an AD orDDD sequence for producing a dock-and-lock (DNL) construct as discussedbelow.

Vaccination with Xenoantigens to Break Tolerance

Novel approaches to anti-cancer vaccine development are summarized inFIG. 1. The discovery in 1996 that a single T-cell receptor canproductively recognize a large continuum of related ligands (Kersh andAllen, 1996, J. Exp. Med. 184:1259-1268; Boesteanu et al., 1998, J.Immunol. 161:4719-4727) has raised the possibility that T cellsrecognizing a xenoantigen may cross-react with its self-homologouscounterpart. In the case of TAA, the autologous T cells specific to TAAsmay have largely been deleted, but T cells specific to the xenoantigeniccounterparts of TAAs may survive the negative selection. These naïve,negative selection-survived, and xenoantigen-specific, T cells, onceinduced and activated by immunization with a xenoantigen, maycross-react with their cognate TAA, due to the plasticity of TCRrecognition. It is therefore an approach to overcome the immunetolerance against homologous self-Ags by immunization with thexenoantigens.

This concept has been verified by the accumulated evidence thatimmunization with xenoantigens is effective in the induction of bothcellular and humoral immune responses against their self counterparts.In a phase I clinical trial, eleven of 21 prostate cancer patientsimmunized with dendritic cells pulsed with recombinant mouse prostaticacid phosphatase developed type-1 T-cell proliferative responses to thehomologous self-Ags, and 6 patients had clinical stabilization of theirpreviously progressing prostate cancer (Fong et al., 2001, J. Immunol.167:7150-7156). In a mouse model of B-cell lymphoma, immunization withxenogeneic human CD20 DNA or its extracellular domain elicits bothT-cell and antibody responses against cells expressing mouse CD20(Roberts et al., 2002, Blood 99:3748-3755; Palomba et al., 2005, Clin.Cancer Res. 11:370-379). Long-term survival was observed in 10-30% miceimmunized with human CD20 extracellular domain DNA, and tumor rejectionwas shown to require CD8⁺ T cells during the effector phase (Palomba etal., 2005, Clin. Cancer Res. 11:370-379).

In another study, C57BL/6 mice, which are tolerant togp75/tyrosinase-related protein-1 (TRP-1), generated autoantibodiesagainst gp75 after immunization with DNA encoding human gp75 but notsyngeneic mouse gp75, resulting in significant tumor protection and therejection of tumor challenge requiring CD4⁺ and NK1.1⁺ cells and Fcreceptor gamma-chain (Weber et al., 1998, J. Clin. Invest.102:1258-1264). In the B16F10LM3 mouse model of melanoma, xenogeneic DNAimmunization with human TRP-2 was effective in protecting mice fromintradermal tumor challenge by a mechanism requiring CD4⁺ and CD8⁺ Tcells. Although this immunization strategy failed to inhibit the growthof established tumors, it prevented local recurrence and the developmentof metastases in the mouse model of minimal residual disease of melanoma(Hawkins et al., 2002, J. Surg. Res. 102(2):137-143). Lu et al. reportedthat a DNA vaccine encoding the extracellular domain of xenogeneic(human) EGFR effectively elicited both protective and therapeuticantitumor immunity against mouse EGFR⁺ lung cancer, indicating theautoimmune response against EGFR⁺ cancer could be induced in across-reaction between the xenogeneic homologous and self-EGFR (Lu etal., 2003, J. Immunol. 170:3162-3170).

In another mouse tumor model, mammary carcinoma in HER-2/neu transgenicmice was effectively inhibited by xenogeneic DNA vaccination (Pupa etal., 2005, Cancer Res. 65:1071-1078; Gallo et al., 2005, Int. J. Cancer113:67-77). More recently, Ko et al. (2007, Cancer Res. 67:7477-7486)reported that self-tolerance in the same mouse tumor model could beefficiently broken by immunization with DNA plasmid and/or adenoviralvector expressing the extracellular and transmembrane domain ofxenogenic human Her-2/neu in combination with gemcitabine therapy.Furthermore, vaccination of HLA-A*0201 transgenic (HHD) mice with humanHer-2(9₄₃₅), the xenogeneic altered peptide ligand of its mousehomologue, significantly increased the frequency of murineHer-2(9₄₃₅)-specific CTL, and also induced strong protective andtherapeutic immunity against the transplantable ALC tumor cell linetransfected to coexpress HLA-A*0201 and hHer-2/neu or rHer-2/neu(Gritzapis et al., 2006, Cancer Res. 66(10):5452-5460).

Of note, it was reported that the elevated frequencies of self-reactiveCD8⁺ T cells following xeno-immunization are due to the presence of aheteroclitic CD4⁺ T-cell helper epitope in the xenoantigen (Kianizad etal., 2007, Cancer Res. 67:6459-6467). In that study, both mDCT(self-antigen) and hDCT (xenoantigen) efficiently elicited specific CD8+and CD⁴⁺ T cells in DCT-deficient mice, whereas in wild-type mice, onlyhDCT elicited a significant level of specific CD8⁺ and CD4⁺ T cells.After introduction of an hDCT-derived dominant CD4⁺ T-cell epitope intomDCT, the mutated mDCT, unlike the native mDCT, elicited potent CD8⁺T-cell frequencies and protective immunity that were comparable to thatwith hDCT. These results suggest a mechanism of action ofxenoimmunization by which xenoantigens can provide heteroclitic CD4⁺helper T-cell epitopes to augment CD8⁺ T-cell immunity against conservedCD8⁺ T-cell epitopes (Kianizad et al., 2007, Cancer Res. 67:6459-6467).Taken together, these studies demonstrate that immunization withxenoantigens is an effective approach to break immune tolerance ofcancer.

Vaccination with Chemically-Modified or Mutated Epitopes

Another approach to break immune tolerance to self-Ags is achieved withchemical modification of antigens (FIG. 1). As early as in 1965, Weiglereported that rabbits immunized with a diazonium derivative-labeledrabbit thyroglobulin produced cross-reactive antibodies to nativethyroglobulin, possibly due to the chemical modification of antigen thatresults in immunogenic epitopes for the cross-reactive antibodyresponses (Weigle, 1965, J. Exp. Med. 121:289-308). Recently, Grunewaldet al. reported that self-tolerance can be overcome by site-specificincorporation of an immunogenic unnatural amino acid into a protein ofinterest to produce high-titer antibodies that cross-react with thewild-type protein (Grunewald et al., 2008, Proc. Natl. Acad. Sci. U.S.A.105:11276-80). Specifically, mutation of a single tyrosine residue(Tyr86) of mTNF-α to p-nitrophenylalanine (pNO2Phe) induced a high-titerantibody response that was highly cross-reactive with native mTNF-α andprotected mice against lipopolysaccharide (LPS)-induced death. Thisstrategy may be applied to the modification and creation of T-cellepitopes.

Other approaches, such as anchor modification of the non-canonicaltumor-associated mucin 1-8 peptide, resulted in enhanced majorhistocompatibility complex class I binding and immune responses (Lazouraet al., 2006, Immunology 119:306-316). The continuously revealedbio-information on the complex of MHC/epitope/TCR and computer modelingof their structures could provide more clues in designing alteredligands/epitopes for induction of efficient immunity againstself-antigens.

Vaccination with Antigens to Induce Tumor-Specific T-Helper Response

Antigen-specific T-helper (Th) cells play a key role in priming,maintaining, and boosting CTL responses (Kennedy, 2008, Immunol. Rev.222:129-144). These Th cells, upon activation at the tumor site byantigen-processing cells (APCs) expressing tumor antigens, provide localor direct growth and survival signals to the tumor-specific CTLs.Strikingly, addition of a tumor-specific Th epitope to a CTL peptidevaccine (OVA257-263, SIINFEKL (SEQ ID NO: 35)) led to an increase insurvival, whereas addition of an irrelevant Th epitope did not, eventhough the addition of either Th epitope to the vaccine resulted ingreater CTL activity (Kennedy and Celis, 2006, J. Immunol.177:2862-2872). Since non-specific Th cells had no beneficial effect onsurvival, it seems that tumor-specific CD4⁺ Th cells directly providehelp at the tumor sites.

Further analysis of this “antigen-specific benefit” in an in vitro modelrevealed that the Th cells protect CTLs from activation-induced celldeath (AICD), which allows CTLs to survive and to continue to kill tumorcells (Kennedy and Celis, 2006, J. Immunol. 177:2862-2872). This modelis supported by the facts that CD4⁺ T cells are required for secondaryexpansion and memory in CD8⁺ T cells (Janssen et al., 2003, Nature421:852-856; Shedlock and Shen, 2003, Science 300:337-339; Sun andBevan, 2003, Science 300:339-342), and that adoptive transfer ofgene-engineered CD4⁺ Th cells induces potent primary and secondary tumorrejections (Moeller et al., 2005, Blood 106(9):2995-3003). It is alsosupported by studies in infectious diseases,

where HIV-specific CD4⁺ T cells are essential for the maintenance ofeffective CTL responses and the generation of functional CTL memorycells (Lichterfeld et al., 2004, J. Exp. Med. 200: 701-712; Kavanach etal., 2006, Blood 107:1963-1969), and are associated with the low HIVviremia in long-term non-progressors (Boar et al., 2002, J. Immunol.169:6376-6385).

The MHC class II-associated invariant chain (Ii), or CD74, acts as achaperone for Ag presentation in the context of MHC class II (Stein etal., 2007, Clin. Cancer Res. 13:5556s-5563s; Matza et al., 2003, TrendsImmunol. 24:264-268). Fusion of antigen to Ii has been shown to increasethe priming of antigen-specific CD4⁺ T cells in vitro and in vivo(Diebold et al., 2008, J. Immunol. 180:3339-3346; Rowe et al., 2006,Mol. Ther. 13:310-319). Furthermore, an adenovirus-encoded vaccinetethered to Ii resulted in a dramatically improved cellular immunitywith augmented presentation of MHC class I-restricted epitopes (Holst etal., 2008, J. Immunol. 180:3339-3346; Grujic et al., 2009, J. Gen.Virol. 90:414-422). Thus, linking antigens to Ii may provide a strategyfor improved vaccination (FIG. 1).

Harnessing DCs to Break Tolerance

Numerous studies have been performed using DCs, the professional andmost potent APCs, to create both preventive and therapeutic vaccinesagainst cancer and infectious diseases, with proven efficacy in animalmodels and patients. However, significant clinical benefit has notpreviously been achieved with DC-based immunotherapy. Recent advances inDC vaccination, which can be achieved either ex vivo or in vivo, are nowbeing evaluated in clinical trials.

Vaccination with Mature DC Generated Ex Vivo

Immature DCs differ from mature DCs not only in the lower T-cellstimulatory capacity due to a low level of MHC class I/II andcostimulatory molecules, but also in their lower capacity of migration.Mature DCs induce T-cell immunity, whereas immature DCs inducetolerance. A DC-based cancer vaccine thus requires fully mature DCs foreffective induction of functionally specific T cells against tumors.Since CD4⁺ T cells provide T-cell help for generating and augmentingtumor-specific CTL responses, and Th1 effector cells play a key role inmediating cellular immunity, DCs that can skew the differentiation ofnaïve CD4⁺ T cells toward Th1 cells, which are termed type-1 DCs (DC1),are preferred for DC-based vaccines.

The in vitro generation of DC1 is largely dependent on the stimulationfactors that induce DC maturation. It was reported that stimulation ofbone marrow-derived murine dendritic cell populations with poly(I:C) andCpGs results in phenotypic maturation of dendritic cells toward DC1,which are characterized as a durable and high-level IL-12p70 secretionand induction of Th1-skewed, tumor-specific, CD4⁺ T-cell response (Hokeyet al., 2005, Cancer Res. 65(21):10059-10067). Polarized DC1 thatproduces high levels of IL-12 family members can rescue patient Th1-typeanti-melanoma CD4⁺ T cell responses in vitro (Wesa et al., 2007, J.Immunother. 30(1):75-82).

Furthermore, a unique protocol (Mailliard et al., 2004, Cancer Res.64:5934-5937) was developed to generate fully mature type-1 human DCs(αDC1) using an optimized maturation cocktail comprising IL-1-β, TNF-α,IFN-α, IFN-γ, and poly(I:C). It was shown that, compared with standardDCs matured by IL-1-β, TNF-α, IL-6, and PGE2, αDC1 expressed similarlevels of multiple costimulatory molecules (CD83, CD86, CD80, CD11c, andCD40), but secreted 10 to 60 times more IL-12p70, and induced muchhigher numbers of functional CD8⁺ T cells against CLL cells, indicatingthis type of DC is the potent inducer of tumor-specific T cells (Lee etal., 2008, J. Leukoc. Biol. 84(1):319-325). In addition, αDC1 potentlyrecruits and activates NK cells (Gustafsson et al., 2008, Cancer Res.68(14):5965-5971). Unlike standard DCs (sDC) generated in the presenceof PGE2, αDC1 lacks the ability of attracting Tregs (Muthuswamy et al.,2008, Cancer Res. 68: 5972-5978). Analysis of the αDC1 maturationcocktail indicated that IFN-γ is the key player for priming DCs toproduce high-levels of IL-12 (Mailliard et al., 2004, Cancer Res.64:5934-5937) and CXCL9/MIG (Gustafsson et al., 2008, Cancer Res.68(14):5965-5971), and IFN-α is a potent inducer of CCR-7 expression,which is essential for efficient migration of DCs to secondary lymphoidorgans (Parlato et al., 2001, Blood 98:3022-3029; Mohty et al., 2003, J.Immunol. 171:3385-3393; Papewalis et al., 2008, J. Immunol.180:1462-1470).

Taken together, given the ability to generate DCs with full maturation,acquisition of CCR-7 expression, Th1 polarization and potent inductionof tumor-specific CTLs, this type of maturation cocktail is likely to beused for developing a new generation of ex vivo manipulated DCs forfuture clinical trials (FIG. 1), even though it was recently reportedthat higher up-regulation of inhibitory molecules, such as PD-L1, ILT2,and ILT3, was found in αDC1 as compared to sDC when generated in aclinical protocol using X-VIVO15. However, αDC1 still consistentlysecreted more IL-12p70 and IL-23 than sDCs (Trepiakas et al., 2008,Vaccine 26:2824-2832).

A further modification of DCs to enhance their T-cell stimulatorycapacity involves the use of lentiviral vectors to engineer theexpression of calnexin (Kang et al., 2002, J. Biol. Chem.277:44838-44844), which converts tolerogenic DCs into reactive DCs inmultiple myeloma and effectively overcomes immune tolerance (Han et al.,2008, Mol. Ther. 16:269-279) (FIG. 1).

Vaccination with DC-Derived Exosomes

Exosomes are 30- to 100-nm diameter vesicles derived from a diverserange of cell types. DC-derived exosomes contain Ag-presenting,adhesion, and costimulatory molecules, which alone or in associationwith DCs can serve as a potent vaccine to stimulate strong CTL responsesand induce antitumor immunity in different animal models (Chaput et al.,2004, J. Immunol. 172:2137-2146; Cho et al., 2005, Int. J. Cancer114:613-622; Hao et al., 2007, Immunology 120:90-102).

For example, mature DCs pulsed with exosomes stimulate efficient CTLresponses and antitumor immunity (Hao et al., 2007, Immunology120:90-102), and active CD4⁺ T cells following uptake of OVA-pulsed,DC-derived exosomes, can stimulate CD8⁺ T cell to proliferate anddifferentiate into central memory CD8⁺ CTLs, resulting in not only moreefficient in vivo antitumor immunity and long-term CD8⁺ T-cell memoryresponses than OVA-pulsed dendritic cells, but also the ability tocounteract CD4⁺25⁺ regulatory T-cell-mediated immune suppression (Hao etal., 2007, J. Immunol. 179:2731-2740). Thus, such exosome-targetedactive CD4⁺ T cells may represent a novel and highly effective cancervaccine (FIG. 1).

Vaccination with In Vivo Targeted DC

In vivo targeting of antigens to DCs represents a promising approach forDC-based vaccination, as it can bypass the laborious and expensiveex-vivo antigen loading and culturing, facilitating large-scaleapplication of DC-based immunotherapy (Tacken et al., 2007, Nat. Rev.Immunol. 7:790-802). More importantly, in vivo DC-targeted vaccinationwas reported to be more efficient in eliciting an anti-tumor immuneresponse, and more effective in controlling tumor growth in animalmodels (Bonifaz et al., 2004, J. Exp. Med. 199:815-824; Kretz-Rommel etal., 2007, J. Immunother. 30:715-726).

One promising strategy developed for in-vivo targeted DC vaccination isto use engineered lentiviral vectors that specifically bind to the DCsurface protein, DC-SIGN (Yang et al., 2008, Nat. Biotechnol.26:326-334). Successful transduction of DCs in vivo by direct injectionof a lentiviral vector encoding the human melanoma antigen, NY-ESO-1,under mouse dectin-2 gene promoter that restricted transgene expressionto antigen-presenting cells, was also evidenced (Lopes et al., 2008, J.Virol. 82:86-95) by priming an NY-ESO-1-specific CD8⁺ T-cell response inHLA-A2 transgenic mice, and stimulating a CD4⁺ T-cell response to anewly identified NY-ESO-1 epitope presented by H2 I-A(b). These resultsindicate that targeting antigen expression to DCs with lentiviralvectors can provide a safe and effective vaccine (FIG. 1).

Targeting antigens to DCs via an antibody specific to a select DC cellsurface marker is another approach for in-vivo targeted DC vaccination,as reported for mannose receptor (He et al., 2007, J. Immunol.178:6259-6267; Ramakrishna et al., 2004, J. Immunol. 172(5), 2845-2852),CD205 (Trumpfheller et al., 2006, J. Exp. Med. 203:607-617; Gurer etal., 2008, Blood 112:1231-1239), DC-SIGN (Tacken et al., 2005, Blood106:1278-1285), and LOX1 (Delneste et al., 2002, Immunity17(3):353-362). In addition, the potential use of CD74 for in-vivotargeted DC vaccination is being explored by us. CD74 is a type-IIintegral membrane protein essential for proper MHC II folding and MHCII-CD74 complex targeting to endosomes (Stein et al., 2007, Clin. CancerRes. 13:5556s-5563s; Matza et al., 2003, Trends Immunol. 24:264-268).CD74 expression is not restricted to DCs, but is in almost allantigen-presenting cells (Freudenthal et al., 1990, Proc. Natl. Acad.Sci. U.S.A. 87:7698-7702), including B cells, monocytes, and differentDC subsets, such as blood myeloid DC1, myeloid DC2, plasmacytoid DC(Chen et al., 2008, Blood (ASH Annual Meeting Abstracts) 112: Abstract2649), and follicular DCs (Clark et al., 1992, J. Immunol.148:3327-3335).

The broad expression of CD74 in APCs may offer some advantages over soleexpression in myeloid DCs, because targeting of antigens to other APCs,like B cells, has been reported to break immune tolerance (Ding et al.,2008, Blood 112:2817-2825), and targeting to plasmacytoid DCscross-presents antigens to naïve CD8 T cells (Mouries et al., 2008,Blood 112:3713-3722). Furthermore, CD74 is also expressed in follicularDCs, a DC subset critical for antigen presentation to B cells (Clark etal., 1992, J. Immunol. 148:3327-3335).

The humanized anti-CD74 monoclonal antibody, hLL1 or milatuzumab (Leunget al., 1995, Mol. Immunol. 32:1416-1427; Losman et al., 1997, Cancer80(12 Suppl):2660-2666; Stein et al., 2004, Blood 104:3705-3711), is atherapeutic MAb currently under clinical evaluation for non-Hodgkinlymphoma, chronic lymphocytic leukemia, and multiple myeloma.Milatuzumab binds efficiently to different subsets of blood DCs, Bcells, monocytes, and monocyte-derived immature DCs, but has nocytotoxicity, nor functional alteration, on human monocyte-derived DCsthat normally express CD74 (Chen et al., 2008, Blood (ASH Annual MeetingAbstracts) 112: Abstract 2649). These properties of milatuzumab, whichinternalizes rapidly upon engagement with CD74, favor its use as aDC-targeting antibody for in vivo vaccination.

Vaccination Against Cancer Stem Cells

Cancer stem cells (CSCs) are capable of self-renewal, possess theability for unlimited proliferation, and are resistant to multipletherapeutic approaches, whereas most mature cancer cells can beeliminated effectively by current standard therapies. A pressingquestion is whether cancer stem cells are sensitive to immunotherapy orvaccination. In the case of leukemia, it was reported that CD8⁺ minorhistocompatibility antigen-specific cytotoxic T lymphocyte clones couldeliminate human acute myeloid leukemia stem cells (Bonnet et al., 1999,Proc. Natl. Acad. Sci. U.S.A. 96:8639-8644). More recently, Rosinski etal. reported that DDX36-encoded H—Y epitope is expressed by leukemicstem cells and can be recognized by the DDX36-specific CTLs, which canprevent engraftment of human acute leukemia in NOD/SCID mice (Rosinskiet al., 2008, Blood 111:4817-4826). Another report demonstrated thatengraftment of mHA myeloid leukemia stem cells in NOD/SCIDγc^(null) micewas completely inhibited by in vitro preincubation with the mHA-specificCTL clone (Kawase et al., 2007, Blood 110:1055-1063). These resultspredict the prospects that immunotherapy would be a potentiallyeffective approach for selective elimination of cancer stem cells, whichcould lead to long-term control of cancer.

However, it is still unknown whether CSCs, like many cancer cells, aresubject to immune tolerance or evasion. CD200, an immunosuppressivemembrane glycoprotein overexpressed in multiple hematologicalmalignancies, and a negative prognostic factor in multiple myeloma(Moreaux et al., 2006, Blood 108:4194-4197) and acute myeloid leukemia(Tonks et al., 2007, Leukemia 21:566-568), has been found to beco-expressed in CSCs with other stem-cell markers in prostate, breast,brain, and colon cancers (Kawasaki et al., 2007, Biochem. Biophys. Res.Commun. 364:778-782). This suggests that CSCs might be able to evadeimmune surveillance or immunotherapy by generating a tolerogenicresponse facilitated by the expression of CD200 (Kawasaki et al., 2008,Trends Immunol. 29:464-468). This fact may increase the difficulty ofeliminating CSCs by immunotherapy approaches. However, it is stillunclear whether CSCs are more resistant to CTL killing than theirprogeny cells, and if so, whether CD200 is a key player for mediatingthe immune resistance.

Cancer Vaccine in Combination with Adoptive Transfer of T Cells

Adoptive transfer of TCR gene-modified T cells is an attractive approachfor the immunotherapy of tumors, especially for those types where it isdifficult or impossible to induce strong T-cell responses byvaccination, and has shown encouraging results in preclinical (Engels etal., 2005, Hum. Gene Ther. 16:799-810; de Witte et al., 2008, J.Immunol. 181:2563-2571; de Witte et al., 2008, J. Immunol.181:5128-5136) and clinical studies (Morgan et al., 2006, Science314:126-129). Moreover, combining vaccination and T-cell adoptivetransfer can exploit the benefits of both modalities. In addition, asadoptive T cells have the lifespan of the host, vaccination can boostthe tumor-specific T cells when needed.

Experimental evidence to support this approach was provided in aspontaneous prostate carcinoma mouse model that whereas vaccination orTCR gene transfer by itself was entirely without effect, the combinationof vaccination with TCR gene transfer was highly synergistic insuppressing tumor development (de Witte et al., J. Immunol.181:2563-2571). Thus, vaccines combined with adoptive T-cell therapycould greatly improve the efficacy of cancer immunotherapy, and wasreported to be the most effective strategy for treating established B 16murine melanoma (Kochenderfer et al., 2007, Exp. Biol. Med. (Maywood)232(9):1130-41) (FIG. 1).

Cancer Vaccine in Combination with Chemotherapy and/or MonoclonalAntibodies

Because of the immunosuppressive effects of cytotoxic therapy, it is achallenge to integrate cancer vaccines into the standard chemotherapy ofcancer. However, chemotherapy or radiotherapy was reported to eliminateregulatory T cells (Tregs) (North et al., 1986, J. Exp. Med.164:1652-1666; Awwad et al., 1989, Cancer Res. 49:1649-1654; Ercolini etal., 2005, J. Exp. Med. 201:1591-1602), which would potentially offersome synergistic action with vaccine-induced anti-tumor effects. Thishas been confirmed by several clinical trials showing that vaccinatedpatients receiving subsequent chemotherapy exhibited better outcomes(longer post-chemotherapy recurrence times and survival) thanvaccination or chemotherapy alone (Baxevanis et al., 2009, CancerImmunol. Immunother. 58:317-324; Wheeler et al., 2004, Clin. Cancer Res.10:5316-5326; Antonia et al., 2006, Clin. Cancer Res. 12:878-887).

Modification of the tumor microenvironment by NSAIDs, such ascyclooxygenase-2 (COX-2) inhibitors, could enhance the clinical efficacyof the cancer vaccine. This is because COX-2 and its downstreamprostaglandins are capable of inhibiting DC and T effector cellactivity, and of stimulating Tregs (Juuti et al., 2006, J. Clin. Pathol.59:382-386; Sharma et al., 2003, Clin. Cancer Res. 9: 961-968; Sharma etal., 2005, Cancer Res. 65:5211-5220; Basu et al., 2006, J. Immunol.177(4): 2391-2402). In a triple transgenic mouse model of spontaneouspancreatic cancer induced by the KRAS^(G12D) mutation and that expresseshuman MUC1 as a self molecule, combination of a MUC1-specific vaccinewith the COX-2 inhibitor, celecoxib, elicited robust antitumor cellularand humoral immune responses, and was associated with increasedapoptosis in the tumor. Strikingly, the immunization was effective onlyin combination with COX-2 inhibition (Mukherjee et al., 2009, J Immunol.182:216-24).

Monoclonal antibodies, either unconjugated or conjugated withradionuclides, are effective in cancer therapy (Sharkey et al., 2006, CACancer J. Clin. 56:226-243; Sharkey et al., 2008, Adv. Drug Deliv. Rev.60:1407-1420). Unconjugated monoclonal antibodies exert their anti-tumorcytotoxicity usually by the mechanisms of complement-mediatedcytotoxicity (CMC) and antibody-dependent cellular cytotoxicity (ADCC),or by initiating or inhibiting signaling pathways in the targeted cellthat leads to apoptosis (Sharkey et al., 2006, CA Cancer J. Clin.56:226-243). Thus, combining vaccine, monoclonal antibody, andchemotherapy may hold more potential for enhancing therapeutic efficacy(Baxevanis et al., 2009, Cancer Immunol. Immunother. 58:317-324) (FIG.1).

Vaccination in Combination with Stem-Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT),following high-dose chemotherapy and total body irradiation, provides aneffective and potentially curative therapy for hematologic malignancies.It is believed that the clinical effectiveness of this approach isprimarily due to the graft versus leukemia (GVL) effect, in which therecipient leukemia cells are recognized and eliminated by donor T cells.The primary target antigen for allo-HSCT with HLA-identical donors isminor-histocompatibility antigen (mHA), which is derived from geneticpolymorphisms in the recipient that are not present in the donor (Ofranet al., 2008, Clin. Cancer Res. 14:4997-4999; Tykodi et al., 2008, Clin.Cancer Res. 14(16):5260-5269). Due to the slow and incomplete immunereconstitution following HSCT, immunization in HSCT is frequentlyunsuccessful (Aqui et al., 2008, Best Pract. Res. Clin. Haematol.21:503-519).

However, effective antitumor immunity was reported to be elicited bytumor vaccination after allo-HSCT in murine models (Anderson et al.,2000, Blood 95:2426-2433; Teshima et al., 2001, Cancer Res. 61:162-171;Moyer et al., 2006, Biol. Blood Marrow Transplant. 12:1010-1019).Recently, it was reported in a clinical study (Kitawaki et al., 2008,Am. J. Hematol. 83:315-317) that DC vaccination with Wilms' tumor 1(WT1) peptide and keyhole limpet hemocyanin (KLH) after allo-HSCTsuccessfully induced immune responses to the nave antigen KLH, eventhough a definitive immune response to WT1 was not detected. This resultindicates that DC vaccination may be a viable strategy forantigen-specific immunotherapy after allo-HSCT (FIG. 1).

Improved Vaccination Schedule to Enhance Anti-Tumor Efficacy

One of the advances in vaccinology is heterologous prime-boost, whichinvolves a sequential vaccination schedule with different antigendelivery systems encoding the same antigen (Hodge et al., 2003, CancerRes. 63:7942-7949; Harrop et al., 2006, Adv. Drug Deliv. Rev.58:931-947). This vaccination schedule was initially designed tocircumvent the problem associated with viral vector-induced neutralizingantibody when given on multiple occasions. In this case, a huge numberof immune cells specific for the viral vector rather than the cellsspecific for the encoded antigen can be expanded, which greatly limitsthe efficacy of vaccination. The use of one viral vector to prime animmune response and a different viral vector to boost the response canminimize the expansion of immune cells specific for vector proteins,which would favor the immune response to the target antigen (Harrop etal., 2006, Adv. Drug Deliv. Rev. 58:931-947).

As for DNA vaccination, a heterologous prime/boost strategy consistingof plasmid-delivered melanoma antigen tyrosinase, followed byrecombinant alphavirus replicon particles encoding the same antigen,resulted in a better immune response and tumor protection thanvaccinating with plasmid DNA alone (Goldberg et al., 2005, Clin. CancerRes. 11:8114-8121). Also, prostate stem cell antigen vaccination inducesa long-term protective immune response without autoimmunity(Garcia-Hernandez et al., 2008, Cancer Res. 68:861-869). The optimalcombination of different vectors and the order in which they should beused to prime and subsequently boost the immune response remains underinvestigation (Harrop et al., 2006, Adv. Drug Deliv. Rev. 58:931-947).Recently, a single heterologous prime-boost trial comparing multiplevaccine vectors identified recombinant vesicular stomatitis virus (rVSV)and recombinant Venezuelan equine encephalitis virus replicons (VRP) asthe most synergistic regimen (Barefoot et al, 2008, Vaccine.26(48):6108-6118).

Xenoantigens

In embodiments involving vaccines against cancer, the vaccines willcomprise a tumor-specific antigen or a tumor-associated antigen. Whilevaccines against tumor-specific antigens provide the greatestselectivity against tumor cells, the need to specifically tailor suchvaccines for the individual patient limits their widespreadapplicability. In contrast, tumor-associated antigens (TAAs), which maybe found to a limited extent in cells of normal tissues, exhibit a muchbroader distribution across tumors from different patients or evendifferent tumor types. In more preferred embodiments, the anti-cancervaccines are targeted to TAAs. A wide variety of TAAs are known in theart and any such known TAA may be used as the basis for an anti-cancervaccine.

In certain preferred embodiments, the TAA is a CD20 xenoantigen, of useto induce an immune response against B cell cancers such as leukemias orlymphomas, or against autoimmune diseases involving B cellproliferation. Other known TAAs include, but are not limited to,carbonic anhydrase IX, alpha-fetoprotein, α-actinin-4, A3, antigenspecific for A33 antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125,CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5,CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25,CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52,CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83,CD95, CD126, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, DAM, EGFR,EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor,G250 antigen, GAGE, gp100, GROB, HLA-DR, HM1.24, human chorionicgonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxia induciblefactor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2,IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15,IL-17, IL-18, IL-25, insulin growth factor-1 (IGF-1), KC4-antigen,KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitoryfactor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP,MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUM-1/2, MUM-3,NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody, placentalgrowth factor, p53, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,ILGF, ILGF-1R, IL-6, IL-25, RS5, RANTES, T101, SAGE, 5100, survivin,survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene markerand an oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207).

Xenoantigen amino acid sequences for a large number of TAAs, such asmurine protein amino acid sequences, may be readily obtained from publicdatabases, such as the NCBI protein database. For example, xenoantigenCD20 amino acid sequences of potential use are readily available to theskilled artisan through such well-known public databases as the NCBIprotein database (see, e.g., NCBI Accession Nos. NP 031667; P19437;AAA37394; BAE47068; ABA29631; BAD77809). Although the murine CD20sequence is recited herein, the skilled artisan will realize that CD20amino acid sequences are known and readily available from a wide varietyof species and can be incorporated into the anti-cancer vaccinecomplexes. Because the xenoantigen amino acid sequence is from adifferent species, the likelihood of self-tolerance of the host immunesystem is substantially reduced.

Dendritic Cell Targeting Antibodies

In certain embodiments, the antigen to be used for vaccine productionmay be targeted to appropriate host cells, such as dendritic cells (DC),by attachment to an appropriate targeting antibody. In a preferredembodiment, the DC-targeting antibody may be an anti-CD74 antibody, suchas the hLL1 antibody (see U.S. Pat. No. 7,312,418, the Examples sectionof which is incorporated herein by reference). However, other antigensassociated with DCs are known, including but not limited to CD209(DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like receptor 2), TLR 4, TLR7, TLR 9, BDCA-2, BDCA-3, BDCA-4, and HLA-DR. Any such known DC antigenmay be targeted using an appropriate antibody vaccine component. Anexemplary anti-HLA-DR antibody is hL243 (see U.S. Pat. No. 7,612,180,the Examples section of which is incorporated herein by reference).

Vaccines for Therapy of Multiple Myeloma and Other Cancers

CD20 is normally expressed in cells of B cell lineage. It was recentlyreported that CD20 is expressed in a small population of MM cellsisolated from MM cell lines or clinical specimens, which do not expressthe characteristic plasma cell surface antigen CD138 but have a highlyclonogenic potential and are resistant to multiple clinical anti-myelomadrugs (Matsui et al., Blood 2004, 103:2332-6; Matsui et al., Cancer Res.2008, 68:190-7). These CD20+CD138-cells are capable of clonogenic growthin vitro and in a 3-D culture model (Kirshner et al., Blood 2008,112:2935-45), and of differentiation into MM cells in vitro and in theengrafted NOD/SCID mouse model during both primary and secondarytransplantation. It has thus been suggested that these CD138^(neg)CD20⁺cells represent the putative multiple myeloma cancer stem cells.

Immunization with Xenoantigen as a Means for Breaking Immune Tolerancefor Cancer Immunotherapy.

Many tumor-associated Ags (TAAs) represent tissue differentiation Agswhich are not inherently immunogenic. T cells that recognize theseTAAs/self-Ags with high avidity are either clonally deleted in thethymus or anergized in the periphery. However, immunization withxenoantigen has been shown to be capable of overcoming the immunetolerance against the homologous self-Ag (Fong et al., J Immunol. 2001,167(12):7150-6). These results demonstrate that xenoantigen immunizationcan break tolerance to a self-Ag in humans, resulting in a clinicallysignificant antitumor effect.

CD20 as a Target for Immunotherapy and Vaccination Against MM.

As stated above, CD20 is a hallmark of MM cancer stem cells. As aself-antigen which is expressed on normal B cells at most stages ofdifferentiation, it is theoretically difficult to be targeted by vaccinestrategies due to immune tolerance. However, successful vaccination hasbeen achieved by a xenogeneic DNA vaccine against CD20 in a tumorchallenge model of B-cell lymphoma. Although autoimmunity against Bcells could be induced by a vaccine targeting CD20, it should not causea large problem because the B cell pool is not a vital and criticaltissue and can be replenished from its lineage progenitor. Based onthese considerations, a therapeutic vaccine targeting CD20 would beeffective in selective eradication of MM cancer stem cells.

Monoclonal Anti-CD20 Antibody as a Potential Modality for Eradication ofMM Stem Cells.

The discovery of CD20+MM progenitor cells has prompted several smallclinical trials to test the efficacy of rituximab, an anti-CD20monoclonal antibody, in MM patients. As reviewed by Kapoor et al. (Br JHaematol. 2008, 141:135-48), anti-CD20 therapy with rituximab elicits apartial response in approximately 10% of CD20+ patients with multiplemyeloma. In addition, there is preliminary evidence of diseasestabilization in 50-57% of CD20+ patients for a period of 10-27 months(Kapoor et al., (Br J Haematol. 2008, 141:135-48). Furthermore, a casereport by Bergua et al. (Leukemia. 2008, 22:1082-3) where rituximab wasused in combination with chemotherapy demonstrated no minimal residualdisease found after treatment, either in immunophenotype, bone marrowaspiration or biopsy, and the CD20+ plasma cells disappeared. Thevaccine approach, due to its induction of CTL response, would beexpected to supplement the monoclonal antibody therapy against CD20 MMstem cells.

In Vivo Targeting of Antigens to Dendritic Cells and OtherAntigen-Presenting Cells as an Efficient Strategy for Vaccination andBreaking Immune Tolerance.

As the professional antigen-presenting cells, dendritic cells (DCs) playa pivotal role in orchestrating innate and adaptive immunity, and havebeen harnessed to create effective vaccines (Vulink et al., Adv CancerRes. 2008, 99:363-407; O'Neill et al., Mol Biotechnol. 2007, 36:131-41).In vivo targeting of antigens to DCs represents a promising approach forDC-based vaccination, as it can bypass the laborious and expensive exvivo antigen loading and culturing, and facilitate large-scaleapplication of DC-based immunotherapy (Tacken et al., Nat Rev Immunol.2007, 7:790-802). More significantly, in vivo DC targeting vaccinationis more efficient in eliciting anti-tumor immune response, and moreeffective in controlling tumor growth in animal models (Kretz-Rommel etal., J Immunother 2007, 30:715-726). In addition to DCs, B cells areanother type of potent antigen-presenting cells capable of primingTh1/Th2 cells (Morris et al, J Immunol. 1994, 152:3777-3785; Constant, JImmunol. 1999, 162:5695-5703) and activating CD8 T cells viacross-presentation (Heit et al., J Immunol. 2004, 172:1501-1507; Yan etal., Int Immunol. 2005, 17:869-773). It was recently reported that invivo targeting of antigens to B cells breaks immune tolerance of MUC1(Ding et al., Blood 2008, 112:2817-25).

CD74 as a Potential Receptor for Targeting Vaccination.

Some receptors expressed on DCs have been used as the targets for invivo antigen targeting, such as the mannose receptor (He et al., J.Immunol 2007, 178, 6259-6267; Ramakrishna et al., J. Immunol. 2004, 172,2845-2852) CD205 (Bonifaz et al., J Exp Med. 2004, 199:815-24), DC-SIGN(Tacken et al., Blood 2005, 106:1278-85), and LOX1 (Deineste et al.,Immunity 2002, 17, 353-362), etc. CD74 is a type II integral membraneprotein essential for proper MHC II folding and targeting of MHC II-CD74complex to the endosomes (Stein et al., Clin Cancer Res. 2007,13:5556s-5563s; Matza et al., Trends Immunol. 2003, 24(5):264-8). CD74expression is not restricted to DCs, but is found in almost allantigen-presenting cells (Freudenthal et al., Proc Natl Acad Sci USA.1990, 87:7698-702; Clark et al., J Immunol. 1992, 148(11):3327-35). Thewide expression of CD74 in APCs may offer some advantages over soleexpression in myeloid DCs, as targeting of antigens to other APCs like Bcells has been reported to break immune tolerance (Ding et al., Blood2008, 112:2817-25), and targeting to plasmacytoid DCs cross-presentsantigens to naïve CD8 T cells. More importantly, CD74 is also expressedin follicular DCs (Clark et al., J Immunol. 1992, 148(11):3327-35), a DCsubset critical for antigen presentation to B cells (Tew et al., ImmunolRev. 1997, 156:39-52). This expression profile makes CD74 an excellentcandidate for in vivo targeting vaccination.

Humanized Anti-CD74 Monoclonal Antibody hLL1 as a Novel Targeting Toolwith Dock-and-Lock Technology Platform.

The DNL technology, discussed in more detail below, provides a means tolink virtually any selected effector moieties into a covalent ornoncovalent complex (Goldenberg et al., J Nucl Med. 2008, 49:158-63;Rossi et al., Proc Natl Acad Sci USA. 2006, 103(18):6841-6). The DNLmethod has generated several trivalent, bispecific, binding proteinscontaining Fab fragments reacting with carcinoembryonic antigen (CEA),and has been successfully used in improved cancer imaging andradioimmunotherapy through a pretargeting strategy (Goldenberg et al., JNucl Med. 2008, 49:158-63).

hLL1 is a humanized monoclonal antibody against human CD74 (Leung etal., Mol Immunol. 1995, 32:1416-1427; Losman et al., Cancer 1997,80:2660-2666; Stein et al., Blood 2004, 104:3705-11). This MAb, in thepresence of cross-linking by a second antibody, exhibits cytotoxicityagainst B cell malignancies. The naked hLL1 is also capable ofcontrolling tumor growth in a MM mouse model. However, our recent datademonstrate that hLL1, in the presence or absence of cross-linking, hasno cytotoxicity against human monocyte-derived DCs. But, our preliminarydata shows hLL1 could efficiently bind different subsets of blood DCsand B cells. It also could moderately induce DC maturation and polarizenaïve T cell differentiation toward Th1 effector cells, suggesting ithas some adjuvant activity and may be a good candidate for use as atargeting tool. This makes it possible and feasible to construct aDNL-based tumor vaccine targeted to APCs through the DNL-carried hLL1antibody.

Immunotherapy for Selective Elimination of Cancer Stem Cells

Cancer stem cells are capable of self-renewal, possess the ability forunlimited proliferation, and are resistant to multiple therapeuticapproaches. In the case of leukemia, it was reported that CD8(+) minorhistocompatibility antigen-specific cytotoxic T lymphocyte clones couldeliminate human acute myeloid leukemia stem cells (Bonnet et al., ProcNatl Acad Sci U.S.A. 1999, 96:8639-8644). As discussed above, otherresults suggest that a vaccine-based immunotherapy approach may beeffective to eliminate cancer stem cells. These results indicate thatimmunotherapy is a potentially effective approach for selectiveelimination of cancer stem cells including MM stem cells, which would berequired for achieving long-term control or even cure of thismalignancy.

Dock and Lock (DNL) Method

In certain embodiments, the vaccine constructs to be prepared and usedmay be made by the novel dock-and-lock (DNL) technique (see, e.g., U.S.Pat. Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143 and 7,666,400, theExamples section of each of which is incorporated herein by reference.)The DNL method is based on the specific protein/protein interactionsbetween the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and the anchoring domain (AD) of A-kinase anchoring proteins(AKAPs) (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell. Biol. 2004; 5: 959). PKA, which plays a centralrole in the signal transduction pathway triggered by the binding of cAMPto the R subunits of PKA, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (R1 and R11), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to theR subunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265:21561).

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci. USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188) and any such known AD sequence may be utilized to forma DNL complex. The amino acid sequences of the AD are quite varied amongindividual AKAPs, with the binding affinities reported for RII dimersranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445). Interestingly, AKAPs will only bind to dimeric R subunits.For human RIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

DDD Of Human RIIα and AD of AKAPs as Linker Modules

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAPs as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a stably tethered structurethrough the introduction of cysteine residues into both the DDD and ADat strategic positions to facilitate the formation of disulfide bonds.The general methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsbring the reactive thiol groups placed onto both the DDD and AD intoproximity (Chimura et al., Proc. Natl. Acad. Sci. USA. 2001; 98:8480) toligate site-specifically.

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,nucleic acids, cytokines and PEG.

DDD and AD Sequence Variants

In certain embodiments, the AD and DDD sequences incorporated into thevaccine complex comprise the amino acid sequences of DDD1 (SEQ ID NO:1)and AD1 (SEQ ID NO:3) below. In more preferred embodiments, the AD andDDD sequences comprise the amino acid sequences of DDD2 (SEQ ID NO:2)and AD2 (SEQ ID NO:4), which are designed to promote disulfide bondformation between the DDD and AD moieties.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

However, in alternative embodiments sequence variants of the AD and/orDDD moieties may be utilized in construction of the vaccine complexes.The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408.)

For example, Kinderman et al. (2006) examined the crystal structure ofthe AD-DDD binding interaction and concluded that the human DDD sequencecontained a number of conserved amino acid residues that were importantin either dimer formation or AKAP binding, underlined below in SEQ IDNO:1. (See FIG. 1 of Kinderman et al., 2006, incorporated herein byreference.) The skilled artisan will realize that in designing sequencevariants of the DDD sequence, one would desirably avoid changing any ofthe underlined residues, while conservative amino acid substitutionsmight be made for residues that are less critical for dimerization andAKAP binding. Thus, a potential alternative DDD sequence of use forconstruction of DNL complexes is shown in SEQ ID NO:5, wherein “X”represents a conservative amino acid substitution. Conservative aminoacid substitutions are discussed in more detail below, but could involvefor example substitution of an aspartate residue for a glutamateresidue, or a leucine or valine residue for an isoleucine residue, etc.Such conservative amino acid substitutions are well known in the art.

Human DDD sequence from protein kinase A (SEQ ID NO: 1)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 5)XXIXIXXXLXXLLXXYXVXVLXXXXXXLVXFXVXYFXXLXXXXX

Alto et al. (2003) performed a bioinformatic analysis of the AD sequenceof various AKAP proteins to design an RII selective AD sequence calledAKAP-IS (SEQ ID NO:3), with a binding constant for DDD of 0.4 nM. TheAKAP-IS sequence was designed as a peptide antagonist of AKAP binding toPKA. Residues in the AKAP-IS sequence where substitutions tended todecrease binding to DDD are underlined in SEQ ID NO:3. Therefore, theskilled artisan will realize that variants which may function for DNLconstructs are indicated by SEQ ID NO:6, where “X” is a conservativeamino acid substitution.

AKAP-IS sequence (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA (SEQ ID NO: 6)XXXXXAXXIVXXAIXXX

Similarly, Gold (2006) utilized crystallography and peptide screening todevelop a SuperAKAP-IS sequence (SEQ ID NO:7), exhibiting a five orderof magnitude higher selectivity for the RII isoform of PKA compared withthe R1 isoform. Underlined residues indicate the positions of amino acidsubstitutions, relative to the AKAP-IS sequence, that increased bindingto the DDD moiety of RIIα. In this sequence, the N-terminal Q residue isnumbered as residue number 4 and the C-terminal A residue is residuenumber 20. Residues where substitutions could be made to affect theaffinity for RIM were residues 8, 11, 15, 16, 18, 19 and 20 (Gold etal., 2006). It is contemplated that in certain alternative embodiments,the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD moietysequence to prepare vaccine constructs. Other alternative sequences thatmight be substituted for the AKAP-IS AD sequence are shown in SEQ IDNO:8-10. Substitutions relative to the AKAP-IS sequence are underlined.It is anticipated that, as with the AKAP-IS sequence shown in SEQ IDNO:3, the AD moiety may also include the additional N-terminal residuescysteine and glycine and C-terminal residues glycine and cysteine, asshown in SEQ ID NO:4.

SuperAKAP-IS (SEQ ID NO: 7) QIEYVAKQIVDYAIHQA Alternative AKAP sequences(SEQ ID NO: 8) QIEYKAKQIVDHAIHQA (SEQ ID NO: 9) QIEYHAKQIVDHAIHQA(SEQ ID NO: 10) QIEYVAKQIVDHAIHQA

Stokka et al. (2006) also developed peptide competitors of AKAP bindingto PKA, shown in SEQ ID NO:11-13. The peptide antagonists weredesignated as Ht31 (SEQ ID NO:11), RIAD (SEQ ID NO:12) and PV-38 (SEQ IDNO:13). The Ht-31 peptide exhibited a greater affinity for the RIIisoform of PKA, while the RIAD and PV-38 showed higher affinity for R1.

Ht31 (SEQ ID NO: 11) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 12) LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 13) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006) developed still other peptide competitors forAKAP binding to PKA, with a binding constant as low as 0.4 nM to the DDDof the RII form of PKA. The sequences of various AKAP antagonisticpeptides is provided in Table 1 of Hundsrucker et al. (incorporatedherein by reference). Residues that were highly conserved among the ADdomains of different AKAP proteins are indicated below by underliningwith reference to the AKAP IS sequence (SEQ ID NO:3). The residues arethe same as observed by Alto et al. (2003), with the addition of theC-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006),incorporated herein by reference.) The sequences of peptide antagonistswith particularly high affinities for the RII DDD sequence are shown inSEQ ID NO:14-16.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AKAP7δ-wt-pep (SEQ ID NO: 14)PEDAELVRLSKRLVENAVLKAVQQY AKAP7δ-L304T-pep (SEQ ID NO: 15)PEDAELVRTSKRLVENAVLKAVQQY AKAP7δ-L308D-pep (SEQ ID NO: 16)PEDAELVRLSKRDVENAVLKAVQQY

Carr et al. (2001) examined the degree of sequence homology betweendifferent AKAP-binding DDD sequences from human and non-human proteinsand identified residues in the DDD sequences that appeared to be themost highly conserved among different DDD moieties. These are indicatedbelow by underlining with reference to the human PKA RIIα DDD sequenceof SEQ ID NO:1. Residues that were particularly conserved are furtherindicated by italics. The residues overlap with, but are not identicalto those suggested by Kinderman et al. (2006) to be important forbinding to AKAP proteins. Thus, a potential DDD sequence is indicated inSEQ ID NO:17, wherein “X” represents a conservative amino acidsubstitution.

(SEQ ID NO: 1) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A (SEQ ID NO: 17) XHIX IP X GL XELLQGYT X EVLR X QP X DLVEFA XX YF XXL XEX R X

The skilled artisan will realize that in general, those amino acidresidues that are highly conserved in the DDD and AD sequences fromdifferent proteins are ones that it may be preferred to remain constantin making amino acid substitutions, while residues that are less highlyconserved may be more easily varied to produce sequence variants of theAD and/or DDD sequences described herein.

In addition to sequence variants of the DDD and/or AD moieties, incertain embodiments it may be preferred to introduce sequence variationsin the antibody moiety or the linker peptide sequence joining theantibody with the AD sequence. In one illustrative example, threepossible variants of fusion protein sequences, are shown in SEQ IDNO:18-20.

(L) (SEQ ID NO: 18) QKSLSLSPGLGSGGGGSGGCG (A) (SEQ ID NO: 19)QKSLSLSPGAGSGGGGSGGCG (-) (SEQ ID NO: 20) QKSLSLSPGGSGGGGSGGCG

Antibodies

In certain embodiments, an antibody or antigen binding fragment thereofmay be incorporated into an anti-cancer vaccine. In preferredembodiments, the antibody binds to a tumor associated antigen (TAA) or aDC-associated antigen. As discussed above variety of tumor-associatedantigens and/or DC-associated antigens are known in the art, andantibodies against any such known antigens may be used.

In other embodiments, antibodies that have a direct therapeutic effecton cancer cells may be used as an adjunct therapy to an anti-cancervaccine. Exemplary anti-cancer antibodies that may be utilized include,but are not limited to, hR1 (anti-IGF-1R, U.S. patent application Ser.No. 12/722,645, filed Mar. 12, 2010) hPAM4 (anti-mucin, U.S. Pat. No.7,282,567), hA20 (anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19,U.S. Pat. No. 7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655),hLL1 (anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat.No. 7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEA, U.S. Pat. No.6,676,924), hMN-15 (anti-CEA, U.S. Pat. No. 7,541,440), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785) and hMN-3 (anti-CEA, U.S. Pat. No.7,541,440) the Examples section of each cited patent or applicationincorporated herein by reference. The skilled artisan will realize thatthis list is not limiting and that any other known anti-cancer antibodymay be used.

In other embodiments, antigen-binding antibody fragments may beutilized. Antigen-binding antibody fragments are well known in the art,such as F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, scFv and the like. As usedherein, an antigen-binding antibody fragment refers to any fragment ofan antibody that binds with the same antigen that is recognized by theintact or parent antibody.

An antibody or fragment thereof which is not conjugated to a therapeuticagent is referred to as a “naked” antibody or fragment. Such nakedantibodies are of use for cancer therapy. In alternative embodiments,antibodies or fragments may be conjugated to one or more therapeutic. Awide variety of such therapeutic are known in the art, as discussed inmore detail below, and any such known therapeutic agent may be usedeither conjugated to an appropriate antibody or unconjugated andadministered before, simultaneously with, or after an anti-cancerantibody and/or vaccine.

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(K) and Y_(R)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Grit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B-cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Imniunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XenoMouse® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody and Fab fragments obtained by disulfidereduction.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies (DABs) are also knownin the art, as disclosed for example in Cossins et al. (2006, ProtExpress Purif 51:253-259), incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Amino Acid Substitutions

In certain embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. In a non-limiting example, the DDDand/or AD sequences used to make the vaccine constructs may be furtheroptimized, for example to increase the DDD-AD binding affinity.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Be (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Therapeutic Agents

In various embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes,radionuclides or other agents may be used as adjunct therapies to thevaccine constructs described herein. Drugs of use may possess apharmaceutical property selected from the group consisting ofantimitotic, antikinase, alkylating, antimetabolite, antibiotic,alkaloid, anti-angiogenic, pro-apoptotic agents and combinationsthereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosurea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vinorelbine,vinblastine, vincristine and vinca alkaloids.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Radionuclides of use include, but are not limited to, ¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb. The therapeutic radionuclidepreferably has a decay-energy in the range of 20 to 6,000 keV,preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter.Maximum decay energies of useful beta-particle-emitting nuclides arepreferably 20-5,000 keV, more preferably 100-4,000 keV, and mostpreferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV. Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O,⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru,¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co,⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like. Someuseful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Te, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA.

In certain preferred embodiments, the therapeutic agent is animmunomodulator. An immunomodulator is an agent that when present,alters, suppresses or stimulates the body's immune system. Such agentsmay be particularly useful in conjunction with vaccines to furthermodulate immune system function. Immunomodulators of use may include acytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β or -γ, and stem cell growth factor, such asthat designated “S1 factor”.

In more preferred embodiments, the effector moieties are cytokines, suchas lymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);placenta growth factor (PlGF), hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor(TNF, such as TNF-α) and LT. In a particularly preferred embodiment, thecytokine is IFN-α2b.

The amino acid sequences of protein or peptide immunomodulators, such ascytokines, are well known in the art and any such known sequences may beused in the practice of the instant invention. The skilled artisan isaware of numerous sources of public information on cytokine sequence.For example, the NCBI database contains both protein and encodingnucleic acid sequences for a large number of cytokines andimmunomodulators, such as erythropoietin (GenBank NM 000799), IL-1 beta(GenPept AAH08678), GM-CSF (GenPept AAA52578), TNF-α (GenPept CAA26669),interferon-alpha (GenPept AAA52716.1), interferon-alpha 2b (GenPeptAAP20099.1) and virtually any of the peptide or protein immunomodulatorslisted above. It is a matter of routine for the skilled artisan toidentify an appropriate amino acid and/or nucleic acid sequence foressentially any protein or peptide effector moiety of interest.Commercial sources of cytokines are also available and may be used, suchas the full-length human IFN-α2b cDNA clone (Invitrogen Ultimate ORFhuman clone cat#HORF01Clone ID IOH35221).

Chemokines of use may include RANTES, MCAF, MW1-alpha, MW1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Conjugation Techniques

In certain embodiments, the antibody or vaccine construct may beconjugated to one or more therapeutic agents. For example, ¹³¹I can beincorporated into a tyrosine of a protein or peptide, or a drug attachedto an epsilon amino group of a lysine residue. Therapeutic agents alsocan be attached, for example to reduced SH groups. Many methods formaking covalent or non-covalent conjugates of therapeutic agents withproteins or peptides are known in the art and any such known method maybe utilized.

A therapeutic agent can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

In some embodiments, a chelating agent may be attached to a protein orpeptide and used to chelate a therapeutic agent, such as a radionuclide.Exemplary chelators include but are not limited to DTPA (such asMx-DTPA), DOTA, TETA, NETA or NOTA. Methods of conjugation and use ofchelating agents to attach metals or other ligands to proteins orpeptides are well known in the art (see, e.g., U.S. Pat. No. 7,563,433,the Examples section of which is incorporated herein by reference).Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs, used with diagnostic isotopes inthe general energy range of 60 to 4,000 keV, such as ¹²⁵I, ¹³¹I, ¹²³I,¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N,¹⁵O or ⁷⁶Br for radioimaging. The same chelates, when complexed withnon-radioactive metals, such as manganese, iron and gadolinium areuseful for MRI. Macrocyclic chelates such as NOTA, DOTA, and TETA are ofuse with a variety of metals and radiometals, most particularly withradionuclides of gallium, yttrium and copper, respectively. Suchmetal-chelate complexes can be made very stable by tailoring the ringsize to the metal of interest. Other ring-type chelates such asmacrocyclic polyethers, which are of interest for stably bindingnuclides, such as ²²³Ra for RAIT are encompassed.

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

A therapeutic agent can be attached at the hinge region of a reducedantibody component via disulfide bond formation. Alternatively, thetherapeutic agent can be conjugated via a carbohydrate moiety in the Fcregion of the antibody. The carbohydrate group can be used to increasethe loading of the same agent that is bound to a thiol group, or thecarbohydrate moiety can be used to bind a different therapeutic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41:832 (1988); Shih et al.,Int. J. Cancer 46:1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313,incorporated herein in their entirety by reference. The general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free aminefunction. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154:5919 (1995); Hansen et al., U.S.Pat. No. 5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868,incorporated herein by reference in their entirety. The engineeredcarbohydrate moiety is used to attach the therapeutic agent.

Methods of Therapeutic Treatment

Various embodiments concern methods of treating a cancer in a subject,such as a mammal, including humans, domestic or companion pets, such asdogs and cats, comprising administering to the subject a therapeuticallyeffective amount of a vaccine construct. The administration of vaccineconstruct can be supplemented by administering concurrently orsequentially a therapeutically effective amount of an antibody thatbinds to or is reactive with an antigen on the surface of the targetcell as discussed above.

The vaccine construct therapy can be further supplemented with theadministration, either concurrently or sequentially, of at least onetherapeutic agent. For example, “CVB” (1.5 g/m² cyclophosphamide,200-400 mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen usedto treat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewell-known to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate, bendamustine, and bryostatin-1.

The vaccine construct can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the vaccineconstruct is combined in a mixture with a pharmaceutically suitableexcipient. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients arewell-known to those in the art. See, for example, Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

The vaccine construct can be formulated for intravenous administrationvia, for example, bolus injection or continuous infusion. Preferably,vaccine construct is infused over a period of less than about 4 hours,and more preferably, over a period of less than about 3 hours. Forexample, the first 25-50 mg could be infused within 30 minutes,preferably even 15 min, and the remainder infused over the next 2-3 hrs.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the vaccine construct. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the vaccine construct. For example, biocompatible polymersinclude matrices of polyethylene-co-vinyl acetate) and matrices of apolyanhydride copolymer of a stearic acid dimer and sebacic acid.Sherwood et al., Bio/Technology 10: 1446 (1992). The rate of releasefrom such a matrix depends upon the molecular weight of the vaccineconstruct, the amount of vaccine construct within the matrix, and thesize of dispersed particles. Saltzman et al., Biophys. J. 55: 163(1989); Sherwood et al., supra. Other solid dosage forms are describedin Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS,5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The vaccine construct may also be administered to a mammalsubcutaneously or even by other parenteral routes. Moreover, theadministration may be by continuous infusion or by single or multipleboluses. Preferably, the vaccine construct is infused over a period ofless than about 4 hours, and more preferably, over a period of less thanabout 3 hours.

More generally, the dosage of an administered vaccine construct forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. The dosage may be repeated as needed, for example, once perweek for 4-10 weeks, once per week for 8 weeks, or once per week for 4weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy. Alternatively, a vaccine construct may beadministered as one dosage every 2 or 3 weeks, repeated for a total ofat least 3 dosages. Or, the construct may be administered twice per weekfor 4-6 weeks. The dosing schedule can optionally be repeated at otherintervals and dosage may be given through various parenteral routes,with appropriate adjustment of the dose and schedule.

In preferred embodiments, the vaccine constructs are of use for therapyof cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, lung cancer including small-cell lung cancer, non-smallcell lung cancer, adenocarcinoma of the lung and squamous carcinoma ofthe lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly foam of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Kits

Various embodiments may concern kits containing components suitable fortreating a disease in a patient. Exemplary kits may contain at least oneor more vaccine constructs as described herein. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used. In certain embodiments, atherapeutic agent may be provided in the form of a prefilled syringe orautoinjection pen containing a sterile, liquid formulation orlyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an anti-cancer vaccine construct, or its constituentfusion proteins. Fusion proteins may comprise an anti-CD74 antibody orCD20 xenoantigen attached to a different peptide or protein, such as theAD and DDD peptides utilized for DNL construct formation as discussed inmore detail in the Examples below. Alternatively the encoded fusionproteins may comprise a DDD or AD moiety attached to a differentantibody or xenoantigen.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci USA 63:78-85 (1969). In other embodiments,an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425, the Examples section of eachincorporated herein by reference.

EXAMPLES

The following examples are provided to illustrate, but not to limit, theclaims of the present invention.

Example 1 Preparation of Dock-and-Lock (DNL) Constructs

Exemplary DNL-vaccine constructs may be formed by combining, forexample, an Fab-DDD fusion protein of an anti-CD74 antibody with aCD20-AD fusion protein. Alternatively, constructs may be made thatcombine IgG-AD fusion proteins with CD20-DDD fusion proteins. Thetechnique is not limiting and any protein or peptide of use may beproduced as an AD or DDD fusion protein for incorporation into a DNLconstruct. Where chemical cross-linking is utilized, the AD and DDDconjugates are not limited to proteins or peptides and may comprise anymolecule that may be cross-linked to an AD or DDD sequence using anycross-linking technique known in the art.

Independent transgenic cell lines may be developed for each DDD or ADfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD-fusion protein module can be combined with any AD-fusion proteinmodule to generate a DNL construct. For different types of constructs,different AD or DDD sequences may be utilized. Exemplary DDD and ADsequences are discussed above.

Expression Vectors

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1). To generate Fab-AD expression vectors, the sequences for thehinge, CH2 and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50. Two shuttle vectorswere designed to facilitate the conversion of IgG-pdHL2 vectors toeither Fab-DDD1 or Fab-AD1 expression vectors, as described below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge (PKSC (SEQ ID NO:21)) followed byfour glycines and a serine, with the final two codons (GS) comprising aBam HI restriction site. The 410 bp PCR amplimer was cloned into thePGEMT® PCR cloning vector (PROMEGA®, Inc.) and clones were screened forinserts in the T7 (5′) orientation.

Construction of (G₄S)₂DDD1 ((G₄S)₂ disclosed as SEQ ID NO:22)

A duplex oligonucleotide, designated (G₄S)₂DDD1 ((G₄S)₂ disclosed as SEQID NO:22), was synthesized by Sigma GENOSYS® (Haverhill, UK) to code forthe amino acid sequence of DDD1 preceded by 11 residues of the linkerpeptide, with the first two codons comprising a BamHI restriction site.A stop codon and an EagI restriction site are appended to the 3′ end.The encoded polypeptide sequence is shown below.

(SEQ ID NO: 23) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, thatoverlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGENOSYS®) and combined to comprise the central 154 base pairs of the 174bp DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase. Following primerextension, the duplex was amplified by PCR. The amplimer was cloned intoPGEMT® and screened for inserts in the T7 (5′) orientation.

Construction of (G₄S)₂-AD1 ((G₄S)₂ disclosed as SEQ ID NO:22)

A duplex oligonucleotide, designated (G₄S)₂-AD1 ((G₄S)₂ disclosed as SEQID NO:22), was synthesized (Sigma GENOSYS®) to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below.

(SEQ ID NO: 24) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the PGEMT® vector and screened for inserts in the T7(5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from PGEMT®with BamHI and NotI restriction enzymes and then ligated into the samesites in CH1-PGEMT® to generate the shuttle vector CH1-DDD1-PGEMT®.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from PGEMT®with BamHI and NotI and then ligated into the same sites in CH1-PGEMT®to generate the shuttle vector CH1-AD1-PGEMT®.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN-14(I)-pdHL2, which has been used to produce hMN-14 IgG, wasconverted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagIrestriction endonucleases to remove the CH1-CH3 domains and insertion ofthe CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above.

Construction of C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:25) and residues 1-13of DDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-PGEMT®,which was prepared by digestion with BamHI and PstI, to generate theshuttle vector CH1-DDD2-PGEMT®. A 507 bp fragment was excised fromCH1-DDD2-PGEMT® with SacII and EagI and ligated with the IgG expressionvector hMN-14(I)-pdHL2, which was prepared by digestion with SacII andEagI. The final expression construct was designatedC-DDD2-Fd-hMN-14-pdHL2. Similar techniques have been utilized togenerated DDD2-fusion proteins of the Fab fragments of a number ofdifferent humanized antibodies.

Construction of h679-Fd-AD2-pdHL2

h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchoring domain sequence of AD2appended to the carboxyl terminal end of the CH1 domain via a 14 aminoacid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD 1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides which comprise the coding sequence forAD2 and part of the linker sequence, were made synthetically. Theoligonucleotides were annealed and phosphorylated with T4 PNK, resultingin overhangs on the 5′ and 3′ ends that are compatible for ligation withDNA digested with the restriction endonucleases BamHI and SpeI,respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-PGEMT®, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-PGEMT®. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Generation of TF2 Trimeric DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). Reducing SDS-PAGE shows that any additional bandsapparent in the non-reducing gel are product-related (not shown), asonly bands representing the constituent polypeptides of TF2 were evident(not shown). However, the relative mobilities of each of the fourpolypeptides were too close to be resolved. MALDI-TOF mass spectrometry(not shown) revealed a single peak of 156,434 Da, which is within 99.5%of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE® assay. TF2,C-DDD1-hMN-14+h679-AD 1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of WI2 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 2 C_(H3)-AD2-IgG Expression Vectors

A plasmid shuttle vector was produced to facilitate the conversion ofany IgG-pdHL2 vector into a C_(H3)-AD2-IgG-pdHL2 vector. The gene forthe Fc (C_(H2) and C_(H3) domains) was amplified by PCR using the pdHL2vector as a template and the following oligonucleotide primers:

Fc BglII Left (SEQ ID NO: 26) AGATCTGGCGCACCTGAACTCCTGFc Bam- EcoRI Right (SEQ ID NO: 27) GAATTCGGATCCTTTACCCGGAGACAGGGAGAG.

The amplimer was cloned in the pGemT PCR cloning vector (Promega). TheFc insert fragment was excised from pGemT with Xba I and Bam HI andligated with AD2-pdHL2 vector that was prepared by digestingh679-Fab-AD2-pdHL2 (Rossi et al., Proc Natl Acad Sci USA 2006,103:6841-6) with Xba I and Bam HI, to generate the shuttle vectorFc-AD2-pdHL2. To convert IgG-pdHL2 expression vectors to aC_(H3)-AD2-IgG-pdHL2 expression vectors, an 861 bp BsrG I/Nde Irestriction fragment was excised from the former and replaced with a 952bp BsrG I/Nde I restriction fragment excised from the Fc-AD2-pdHL2vector. The following is a partial list of C_(H3)-AD2-IgG-pdHL2expression vectors that have been generated and used for the productionof recombinant humanized IgG-AD2 modules:

C_(H3)-AD2-IgG-hA20 (anti-CD20)

C_(H3)-AD2-IgG-hLL2 (anti-CD22)

C_(H3)-AD2-IgG-hL243 (anti-HLA-DR)

C_(H3)-AD2-IgG-hLL1 (anti-CD74)

C_(H3)-AD2-IgG-hR1 (anti-IGF-1R)

C_(H3)-AD2-IgG-h734 (anti-Indium-DTPA).

Example 3 Production of C_(H3)-AD2-IgG

Transfection and Selection of Stable C_(H3)-AD2-IgG Secreting Cell Lines

All cell lines were grown in Hybridoma SFM (Invitrogen, CarlsbadCalif.). C_(H3)-AD2-IgG-pdHL2 vectors (30 μg) were linearized bydigestion with Sal I restriction endonuclease and transfected intoSp2/0-Ag14 (2.8×10⁶ cells) by electroporation (450 volts, 25 μF). ThepdHL2 vector contains the gene for dihydrofolate reductase allowingclonal selection as well as gene amplification with methotrexate (MTX).

Following transfection, the cells were plated in 96-well plates andtransgenic clones were selected in media containing 0.2 μM MTX. Cloneswere screened for C_(H3)-AD2-IgG productivity by a sandwich ELISA using96-well microtitre plates coated with specific anti-idiotype MAbs.Conditioned media from the putative clones were transferred to themicro-plate wells and detection of the fusion protein was accomplishedwith horseradish peroxidase-conjugated goat anti-human IgG F(ab′)₂(Jackson ImmunoResearch Laboratories, West Grove, Pa.). Wells giving thehighest signal were expanded and ultimately used for production.

Production and Purification of C_(H3)-AD2-IgG Modules

For production of the fusion proteins, roller bottle cultures wereseeded at 2×10⁵ cells/ml and incubated in a roller bottle incubator at37° C. under 5% CO₂ until the cell viability dropped below 25% (˜10days). Culture broth was clarified by centrifugation, filtered, andconcentrated up to 50-fold by ultrafiltration. For purification ofC_(H3)-AD2-IgG modules, concentrated supernatant fluid was loaded onto aProtein-A (MAB Select) affinity column. The column was washed tobaseline with PBS and the fusion proteins were eluted with 0.1 MGlycine, pH 2.5.

Example 4 Generation of DDD2-mCD20(136-178) and Construction ofDDD2-mCD20(136-178)-pdHL2

DDD2-mCD20(136-178)-pdHL2 is the expression vector forDDD2-mCD20(136-178), which comprises DDD2-linker-mCD20(136-178)-HHHHHH(HHHHHH disclosed as SEQ ID NO:28). The extracellular domain of mouseCD20 (mCD20) is referred to as mCD20(136-178), comprising the sequenceshown below:

(SEQ ID NO: 29) TLSHFLKMRRLELIQTSKPYVDIYDCEPSNSSEKNSPSTQYCN

The amino acid sequence of mouse CD20 xenoantigen is shown below.

(SEQ ID NO: 30) MSGPFPAEPTKGPLAMQPAPKVNLKRTSSLVGPTQSFFMRESKALGAVQIMNGLFHITLGGLLMIPTGVFAPICLSVWYPLWGGIMYIISGSLLAAAAEKTSRKSLVKAKVIMSSLSLFAAISGIILSIMDILNMTLSHFLKMRRLELIQTSKPYVDIYDCEPSNSSEKNSPSTQYCNSIQSVFLGILSAMLISAFFQKLVTAGIVENEWKRMCTRSKSNVVLLSAGEKNEQTIKMKEEIIELSGVSSQPKNEEEIEIIPVQEEEEEEAEINFPAPPQEQESLPVENEIAP

The DNA segment comprising the nucleotide sequence of mCD20(136-178)flanked by BamHI and XhoI restriction sites is obtained by PCR using afull length murine CD20 cDNA clone as template and the two primers shownbelow:

Upstream primer: BamHI_mCD20 primer (30-mer) (SEQ ID NO: 31) 5′- GGATCCACACTTTCTCATTTTTTAAAAATG Downstream primer: XhoI mCD20 primer (30-mer)(SEQ ID NO: 32) 5′- CTCGAG GTTACAGTACTGTGTAGATGGGGA

The PCR amplimer (141 bp) is cloned into the PGEMT® vector (PROMEGA®). ADDD2-pdHL2 mammalian expression vector, for example,N-DDD2-hG-CSF-His-pdHL2, is prepared for ligation with the amplimer bydigestion with XbaI and Bam HI restriction endonucleases. ThemCD20-amplimer is excised from PGEMT® with XbaI and Bam HI and ligatedinto the DDD2-pdHL2 vector to generate the expression vectorDDD2-mCD20(136-178)-pdHL2.

Transfection and Screen to Obtain Clones Expressing DDD2-mCD20(136-178)

The vector DDD2-mCD20(136-178) is linearized by digestion with SalIenzyme and stably transfected into SpESF myeloma cells byelectroporation (see, e.g., U.S. Pat. No. 7,537,930, the Examplessection of which is incorporated herein by reference). A number ofclones are found to have detectable levels of DDD2-mCD20(136-178) byELISA, from which the best producing clone is selected and subsequentlyamplified with increasing methotrexate (MTX) concentrations from 0.1 to0.8 μM over five weeks. At this stage, it is sub-cloned by limitingdilution and the highest producing sub-clone is expanded.

The clone is expanded to 34 roller bottles containing a total of 20 L ofserum-free Hybridoma SFM with 0.8 μM MTX and allowed to reach terminalculture. The supernatant fluid is clarified by centrifugation andfiltered (0.2 μM). The filtrate is diafiltered into 1× Binding buffer(10 mM imidazole, 0.5 M NaCl, 50 mM NaH₂PO₄, pH 7.5) and concentrated to310 mL in preparation for purification by immobilized metal affinitychromatography (IMAC). The concentrate is loaded onto a 30-mL Ni-NTAcolumn, which is washed with 500 mL of 0.02% Tween 20 in 1× bindingbuffer and then 290 mL of 30 mM imidazole, 0.02% Tween 20, 0.5 M NaCl,50 mM NaH₂PO₄, pH 7.5. The product is eluted with 110 mL of 250 mMimidazole, 0.02% Tween 20, 150 mM NaCl, 50 mM NaH₂PO₄, pH 7.5. Thepurity of DDD2-mCD20(136-178) is assessed by SDS-PAGE under reducingconditions.

Example 5 Generation of 74-mCD20 DNL Vaccine Comprising hLL1 IgG Linkedto Four Copies of mCD20(136-178)

C_(H3)-AD2-IgG-hLL1 (anti-CD74) is produced as described in Examples 2and 3. The construct comprises an AD2 moiety attached to the C-terminalend of each heavy chain of the hLL1 IgG. DDD2-mCD20(136-178) is producedas described in Example 4. A DNL reaction is performed by mixing hLL1IgG-AD2 and DDD2-mCD20(136-178) in PBS containing 1 mM reducedglutathione. On the next day oxidized glutathione is added to a finalconcentration of 2 mM and the reaction mixture is purified on a ProteinA column 24 h later. In this embodiment, two copies of the DDD2-mCD20are attached to each AD2 moiety, resulting in a DNL complex comprisingone hLL1 IgG moiety and four mCD20 xenoantigen moieties.

In an alternative embodiment, the Fab of hLL1 is linked to DDD2 and themCD20(136-178) to AD2. Formation of a DNL construct as described aboveresults in the formation of an MM vaccine, designatedhLL1-F(ab)₂-mCD20(136-178), which comprises a single mCD20(136-178)attached to two Fab moieties of hLL1. The generation ofAD2-mCD20(136-178) is described in Example 6.

Administration of 74-mCD20(136-178) or hLL1-F(ab)₂-mCD20(136-178) tosubjects with MM induces an immune response against CD138^(neg)CD20⁺putative MM stem cells. The immune response is effective to reduce oreliminate MM disease cells in the subjects.

Example 6 Generation of Recombinant AD2-mCD20(136-178)

AD2-mCD20(136-178)-pdHL2 is the expression vector for recombinantAD2-mCD20(136-178), which comprisesAD2-linker-mCD20(136-178)-HHHHHH(HHHHHH disclosed as SEQ ID NO:28). TheDNA segment comprising the nucleotide sequence of mCD20(136-178) flankedby Bgl2 and Eag1 restriction sites is obtained by PCR using a fulllength murine CD20 cDNA clone as template and the two primers shownbelow:

Upstream primer: Bgl2_mCD20 primer (30-mer) (SEQ ID NO: 33) 5′- AGATCTACACTTTCTCATTTTTTAAAAATG Downstream primer: Eag1_mCD20 primer (48-mer)(SEQ ID NO: 34) 5′ CGGCCG TCAGTGGTGGTGGTGGTGGTGGTTACAGTACTGTGTAGATGG

The PCR amplimer (162 bp) is cloned into the PGEMT® vector (PROMEGA®).An AD2-pdHL2 mammalian expression vector, for example,N-AD2-hTransferrin-His-pdHL2, is prepared for ligation with the amplimerby digestion with Bgl2 and Eag1 restriction endonucleases. ThemCD20-amplimer is excised from PGEMT® with Bgl2 and Eag1 and ligatedinto the AD2-pdHL2 vector to generate the expression vectorAD2-mCD20(136-178)-pdHL2. Clones expressing AD2-mCD20(136-178) areobtained as described in Example 4 and AD2-mCD20(136-178) is purifiedfrom culture supernatants using Ni-select.

Example 7 Effects of hLL1 on DCs-Efficient Binding of hLL1 withDifferent Subsets of APCs

Early studies demonstrated that CD74 is expressed in mostantigen-presenting cells including blood DCs, B cells, monocytes. Tofurther characterize the expression profile of CD74 in APCs, we examinedthe expression of CD74 in different subsets of human PBMCs and in vitromonocyte-derived DCs. Using the gating strategy that is shown in FIG.2A, we found all of the blood DC subsets, the myeloid DC1 (MDC1) and DC2(MDC2), and plasmacytoid DC (PDC) expressed CD74, with MDC2 expressingthe highest level of CD74 (FIG. 2B). CD74 was also expressed inmonocyte-derived immature DCs at much higher level than in LPS-maturedDCs (FIG. 3A). Consistent with the CD74 expression profiles, hLL1 boundefficiently with blood DC subsets, B cells, monocytes, andmonocyte-derived immature DCs (FIG. 2C, FIG. 3B), but not LPS-maturedDCs (FIG. 3B, FIG. 3C). The binding efficiency of hLL1 in these APCsubsets correlates well with their CD74 expression levels. These dataprovide the basis for in vivo targeting of antigen to APCs using hLL1 asthe targeting vehicle by Dock-and-lock technology.

Cytotoxic Effect of hLL1 on CD74-Expressing Malignant B Cells but not onNormal DCs

Since CD74 is highly expressed in immature DCs, with which hLL1 bindsefficiently, as shown in FIG. 2A and FIG. 2B, we wondered if hLL1 hasthe same cytotoxicity in DCs, as it does a in CD74-expressing B celllymphoma, which was shown previously (Stein et al., Blood 2004,104:3705-11). To this end, the effects of hLL1 on the cell viability ofB cell malignancy Daudi cells and human monocyte-derived DCs wereside-by-side compared using an MTS assay and microscope imaging. Theresults demonstrated that hLL1, in the presence of GAH (goat anti-humanantibody), the second antibody for hLL1 cross-linking, significantlyreduced cell viability of Daudi cells but not DCs (FIG. 4A), whichnormally expressed high level of CD74 as shown above. The microscopicimaging showed that Daudi cells treated with hLL1 crosslinked with GAHbecame clumped and condensed, while the DCs maintained normal morphologyafter the same treatment (FIG. 4C, FIG. 4D). The cytotoxicity againstDaudi cells by hLL1 cross-linked with GAH was consistent with theearlier study by Stein et al. (2004) showing that hLL1 was cytotoxic toB cell malignancies in vitro and in vivo. The lack of cytotoxicity ofhLL1 plus GAH on DCs was further demonstrated in apoptosis assay, whichshowed that the hypodiploid nuclei populations were not influenced byhLL1 cross-linked with GAH (not shown).

To further confirm the lack of cytotoxicity of hLL1 on DCs, we performedapoptosis assay using flow cytometry. The nuclei from hLL1 treatedimmature DCs were obtained and stained with PI for flow cytometryanalysis. The PI+ particles were gated first, and the debris wasexcluded by gating out the SSC-low particles. The resulting gated nucleiwere analyzed for apoptosis by measuring hypodiploid nuclei population(FIG. 3A). The results demonstrate that hLL1 had no influence on DCapoptosis in both donors (FIG. 3B, FIG. 3C), in the presence or absenceof a second mAb (20 μg/ml) for cross-linking (GAH, F(ab′)₂ GAH IgGFcγ-specific). These data demonstrated that hLL1, unlike its cytotoxiceffect on B cell malignancies, has little cytotoxicity against normaldendritic cells which also express CD74 surface antigen.

Moderate Enhancement of DC Constitutive Maturation by hLL1

Human IgG can interact with DCs through FcR ligation and has opposingeffects on DC maturation depending on which subtype(s) of FcR isinvolved. hLL1, as a humanized IgG, may interact with human DCs not onlythrough CD74 but also through FcR expressed on DCs. For this reason, wespeculated that hLL1 may influence DC functions through interaction withCD74 or FcR, or both. To investigate this, we tested the effect of hLL1on DC constitutive maturation during in vitro culture of monocytes inthe presence of hGM-CSF and hIL-4.

Since DC maturation is usually reflected by its morphological change, wealso examined if hLL1 treatment has any effect on DC morphology. Asshown in FIG. 4B, DCs treated with hLL1, at different doses for variousdays, in the absence or presence of GAH cross-linking, appeared healthyand intact. The hLL1-treated DCs exhibited some minor morphologicalchanges featured with fiber-like cells, which are similar to but lessobvious than LPS-treated DCs (not shown).

As mature DCs differ from immature DCs mainly in the upregulation ofantigen-presenting and costimulatory molecule expression, alteredcytokine production, and enhanced T-cell stimulatory ability, we theninvestigated if hLL1 has any effect on the expression level ofantigen-presenting molecule HLA-DR and costimulatory molecules CD54 andCD86 in DCs (FIG. 5). The results show that hLL1 could upregulateHLA-DR, CD54, and CD86 in a dose-dependent manner within the range ofhLL1 concentrations at 0.05-5 ug/ml (FIG. 5A). However, the effect wasnot strong, as the expression of HLA-DR and costimulatory molecules,CD54 and CD86, were only 10% upregulated at 5 μg/ml hLL1 compared to 0ug/ml (FIG. 5B). At the highest concentration (50 μg/ml), the expressionof HLA-DR, CD54 and CD86 was not further upregulated but slightlyreduced, compared to hLL1 at 5 μg/ml (FIG. 5B). These results indicatethat hLL1, although not potently, could enhance the constitutivematuration of DCs.

No Significant Influence on T Cell Expansion by hLL1-Treated DCs

The functional difference between immature DCs and mature DCs is thatmature DCs have a stronger capacity to stimulate T cell proliferationand expansion. Since hLL1 could enhance the constitutive maturation byupregulating the expression of HLA-DR, CD54 and CD86 expression in DCs(FIG. 5B), we determined whether this DC-maturing effect could bereflected by an enhanced T cell expansion by DCs. As shown in FIG. 6,DCs treated with hLL1 at 0.05 to 50 μg/ml did not influence theDC-mediated T cell expansion, including total T cells, CD4+ and CD4− Tcells (FIG. 6). This result suggests that hLL1-enhanced DC constitutivematuration was not strong enough to be translated into an enhanced Tcell stimulatory ability.

Polarization of NaïVe CD4+ T Cells Toward Th1 Effector Cells byhLL1-Treated DCs

However, DCs have another important function: the polarization of naïveCD4 T cells to differentiate into different effector cells, Th1, Th2,Th17, as well as newly defined Th17-1 cells. Th1 cells are critical forcellular immunity against intracellular pathogens and cancers, whereasinduction of Th2 cells is responsible for humoral immunity. TheIL-17-producing Th17 and Th17-1 cells are other polarized cellpopulations which have multiple functions in immunity to certainpathogens and autoimmune inflammation. The polarization of theseeffector cells is largely mediated through DC-secreted cytokines, theso-called “signal 3”, that DCs provide to T cells in the DC/T cellsynapse. The CD4+ naïve T cells can differentiate into Th1, Th2 and Th0cells which mediate different effector functions, among which the Th1effector cells play an essential role in maintaining CTL responseagainst cancer and infectious diseases. We have shown that hLL1 at 0.05to 50 μg/ml could enhance DC constitutive maturation in a weak butdose-dependent manner, but DCs treated with these concentrations of hLL1didn't influence the DC-mediated T cell expansion (FIG. 6). We were theninterested if the hLL1-treated DCs could influence the polarization ofCD4+ naïve T cells. As shown in FIG. 6, hLL1-treated DCs polarized theCD4+ naïve T cells to differentiate toward more Th1 effector cells andfewer Th2 and Tnp cells. These results indicate that DCs can befunctionally modulated by hLL1. As Th1 plays a crucial role in adaptiveimmunity against tumor and infectious diseases, hLL1 may have anadjuvant-like activity when used in vaccination.

Example 8 In Vitro Properties of 74-mCD20—Induction of hCD20-SpecificImmunity by 74-mCD20 in Human PBMCs

CD20 is a self antigen normally expressed on B cells, which istheoretically difficult to target by vaccine strategies due to immunetolerance. However, specific T-cell immune response to CD20 has beenachieved in tumor bearing mice by vaccination with a minigene encodingthe extracellular domain of human CD20 (Palomba et al., Clin Cancer Res2005; 11:370-9), or a conjugate comprising the extracellular domain ofhuman CD20 and a carrier protein with QS21 adjuvant (Roberts et al.,Blood 2002; 99:3748-55). Several other reports have also demonstratedthe feasibility of using xenoantigens to break immune tolerance, asshown for MUC1 in animal models (Ding et al., Blood 2008; 112:2817-25;Soares et al., J Immunol 2001; 166:6555-63) as well as in patients(Ramanathan et al., Cancer Immunol Immunother 2005; 54:254-64). To testwhether 74-mCD20ould successfully induce hCD20-specific immunity andovercome the immune tolerance of CD20, the following experiment isperformed.

Human DCs are generated from PBMCs by culturing for 5 days in thepresence of hGM-CSF and hIL-4. The immature DCs are loaded with74-mCD20, and matured by LPS plus IFN-gamma. The mature DCs are used tostimulate autologous PBMCs for 10 days. Restimulation with the sameloaded DCs is performed twice weekly. After the last restimulation, theT cells are tested for their antigen specificity by measuring cytokineresponse (IFN-gamma) upon stimulation by sorted CD20-positive MM cancerstem cells. The CD20-negative MM cells are used as a control. The Tcells show a positive reaction to CD20-positive MM cancer stem cells butnot to control CD20-negative MM cells.

Specific Binding, Internalization and Intracellular Location of 74-mCD20in Various Antigen-Presenting Cells In Vitro

Our preliminary data have shown that hLL1 efficiently and specificallybinds with different APCs, including myeloid DC1 and myeloid DC2,plasmacytoid DC, B cells and monocytes. In order to confirm that74-mCD20 has the same efficiency and specificity in binding with APCs ashLL1 alone, the following experiment is performed.

74-mCD20 and the control M1-mCD20 (comprising the anti-MUC1 antibodyhPAM4 linked to four copies of mCD20) are used. Binding assays areperformed as follows. Briefly, 15 μg of 74-mCD20 or M1-mCD20 are labeledwith a ZENON™ ALEXA FLUOR® 488 human IgG labeling kit (INVITROGEN®)following the manufacturer's instructions. The labeled preparations areused to stain the human PBMCs as described below.

Human PBMCs isolated from buffy coat using FICOLL-PAQUE™ are treatedwith human FcR blocking Reagent (Miltenyi Biotec, 1:20 dilution) at 4°C. for 10 min. The washed cells are stained with specifically labeledmAbs and analyzed by flow cytometry (FACSCALIBUR®). The labeled mAbsused for the study include FITC-labeled anti-CD74 mAb ALEXA FLUOR®488-labeled 74-mCD20; ALEXA FLUOR® 488-labeled M1-mCD20; PE-conjugatedanti-CD19 mAb (for B cells); PE-conjugated anti-CD14 mAb (formonocytes); and APC-conjugated mAb to BDCA-1 (for MDC1), BDCA-2 (forPDC), or BDCA-3 (for MDC2). A gating strategy is used for identificationof B cells, monocytes, MDC1, MDC2, and PDC. Data were analyzed by FlowJosoftware for mean fluorescence intensity and positive cell populationsexpressing the surface markers.

To see if 74-mCD20 is internalized to endosomes for further processingto MHC class II presentation and MHC class I cross-presentation, thefollowing experiment is performed. 74-mCD20 or M1-mCD20 is mixed withhuman PBMCs, and incubated at 4° C. for 1 hr, followed by extensivewashing. The cells are then transferred to 37° C., fixed at differenttime points (0, 15, 30, or 45 min) and stained with ALEXA FLUOR®-labeledanti-human IgG secondary antibody with or without priorpermeabilization. The mean fluorescence is determined by flow cytometry,and the amount of internalized antibody is calculated by subtracting themean fluorescence in fixed cells (surface bound) from that recorded withfixed and permeabilized cells (internalized and surface bound) atvarious time points.

The results show that the 74-mCD20 DNL complex has the same efficiencyand specificity in binding with APCs as hLL1 alone.

Example 9 Induction of hCD20-Specific Immune Responses by 74-mCD20 InVivo

Intrahepatic injection of CD34+ human cord blood cells (HLA A1 healthydonor) into irradiated newborn Rag2−/−γc−/− mice is performed togenerate the animal model for a reconstituted human adaptive immunesystem including human T, B, and DC cells, and structured primary andsecondary lymphoid organs (Huff et al., J Clin Oncol. 2008, 26:2895-900;Yang and Chang, Cancer Invest. 2008, 26:741-55). These mice are calledHu-Rag2−/−γc−/− mice.

To assess the immune responses induced by 74-mCD20, human CD34+ cellsreconstituted in Rag2−/−γc−/− mice are immunized weekly for three timeswith 74-mCD20 or M¹-mCD20(50 μg per mouse), in combination with orwithout CpG (50 μg per mouse) for in vivo DC maturation. Five days afterthe last immunization, splenocytes of each animal are isolated andrestimulated with HLA-matched MM cancer stem cells for cytokine(IFN-gamma) production, as assessed by intracellular cytokine stainingwith flow cytometry. The specific cytotoxicity against MM cancer stemcells is assessed by a calcein AM release assay with MM cancer stemcells as the target cells. The CD20+MM cancer stem cells are isolatedfrom the MM cell line RPMI18226 using magnetic beads. The stem cellproperty is verified by staining with aldehyde dehydrogenase. Theresults indicate that 74-mCD20 is capable of inducing an anti-hcd20specific immune response in vivo.

Example 10 Therapeutic Potential of 74-mCD20 Against MM Cancer StemCells: In Vivo Evaluation by hPBMC/NOD/SCID Mouse Model or AdoptiveTransfer

The best way for in vivo evaluation of the therapeutic effect of74-mCD20 is to immunize an animal model that can support both the growthof MM and the development of a human adaptive immune system. Since humanCD34+ cell-reconstituted Rag2−/−γc−/− mice are immune-competent, whichmay not support MM growth, the hPBMC/NOD/SCID mouse model is used totest the therapeutic effect of 74-mCD20 against MM stem cells. TheNOD/SCID mice have been used for engraftment of clonogenic multiplemyeloma stem cells by Matsui et al. (Blood 2004, 103:2332-6; Cancer Res2008, 68:190-7).

The NOD/SCID mice are also used for evaluating the therapeutic effect byco-engraftment of tumor cells and hPBMC. By carefully adjusting the cellnumbers infused, this model can support both tumor growth and hPBMCengraftment, and has been used for testing the effect of an in vivovaccine targeting DC-SIGN.

Four to six-week-old female NOD/SCID mice (Jackson Laboratories, BarrHarbor, Me.) are irradiated with 300 cGy (84 cGy/min using a 137Cs gammairradiator). 12-16 h later, sorted CD20+MM cancer stem cells (2 million)are injected via dorsal tail vein. Meanwhile, a mixture of human PBMCs(3 million), immature DC (30,000) and the DNL vaccine is injected intothe mice subcutaneously. At certain time points (days), mice areeuthanatized and bone marrow is harvested from the long bones and theengraftment and therapeutic efficacy are determined by staining forhuman CD138⁺ MM cells.

In order to further evaluate the therapeutic potential of 74-mCD20, analternative method by adoptive transfer is used to test thevaccine-elicited cytotoxicity against MM stem cells. The human CD34+cell-reconstituted Rag2−/−γc−/− mice are immunized with 74-mCD20 asdescribed above. The splenocytes are harvested and injected via the tailvein into NOD/SCID mice engrafted with CD20+MM cancer stem cells. Atcertain time points (days), mice are euthanatized and bone marrow isharvested from the long bones and the engraftment and therapeuticefficacy are determined by staining for human CD 138+MM cells. Theresults confirm that 74-mCD20 is capable of inducing an immune responseagainst CD20⁺ MM stem cells in vivo.

Example 11 Generation of DDD2-mPAP and DNL Vaccine Complex

A DDD2 conjugated PAP xenoantigen is generated from murine prostaticacid phosphatase according to the method of Example 4. The efficacy ofdendritic cell based vaccination with a PAP xenoantigen has beenpreviously disclosed (Fong et al. J Immunol 2001, 167:7150-56). ADDD2-mPAP-pdHL2 expression vector is constructed as described in Example4 and the DDD2-mPAP xenoantigen fusion protein is expressed in cellculture according to Example 4. The murine prostatic acid phosphatasesequence is disclosed, for example, in the NCBI database at AccessionNo. AAF23171. A DDD2-mPAP-6His fusion protein (“6His” disclosed as SEQID NO: 28) is expressed and purified by immobilized metal affinitychromatography (IMAC) as described in Example 4.

A DNL construct comprising one copy of C_(H3)-AD2-IgG-hLL1 (anti-CD74)and four copies of DDD2-mPAP is prepared according to the methods ofExample 5. The hLL1 IgG moiety comprises an AD2 sequence attached to theC-terminal end of each heavy chain of the hLL1 IgG. A DNL reaction isperformed by mixing hLL1 IgG-AD2 and DDD2-mPAP in PBS containing 1 mMreduced glutathione. On the next day oxidized glutathione is added to afinal concentration of 2 mM and the reaction mixture is purified on aProtein A column 24 h later. Two copies of the DDD2-mPAP are attached toeach AD2 moiety, resulting in a DNL complex comprising one hLL1 IgGmoiety and four mPAP xenoantigen moieties.

Administration of DNL vaccine anti-CD74-mPAP to subjects with prostatecancer induces an immune response against PAP expressing prostaticcancer stem cells. The immune response is effective to reduce oreliminate prostatic cancer cells in the subjects.

Example 12 Generation of DDD2-mEGFR and DNL Vaccine Complex

A DDD2 conjugated EGFR xenoantigen is generated from murine EGFRaccording to the method of Example 4. The efficacy of EGFR xenoantigenat inducing a humoral immune response has been previously disclosed(Fang et al. Int J Mol Med 2009, 23:181-88). A DDD2-mEGFR-pdHL2expression vector comprising the extracellular domain of murine EGFR isconstructed as described in Example 4 and the DDD2-mEGFR xenoantigenfusion protein is expressed in cell culture according to Example 4. Themurine EGFR sequence is disclosed, for example, in the NCBI database atAccession No. AAG43241. A DDD2-mEGFR-6His fusion protein (“6His”disclosed as SEQ ID NO: 28) is expressed and purified by immobilizedmetal affinity chromatography (IMAC) as described in Example 4.

A DNL construct comprising one copy of C_(H3)-AD2-IgG-hLL1 (anti-CD74)and four copies of DDD2-mEGFR is prepared according to the methods ofExample 5. The hLL1 IgG moiety comprises an AD2 sequence attached to theC-terminal end of each heavy chain of the hLL1 IgG. A DNL reaction isperformed by mixing hLL1 IgG-AD2 and DDD2-mEGFR in PBS containing 1 mMreduced glutathione. On the next day oxidized glutathione is added to afinal concentration of 2 mM and the reaction mixture is purified on aProtein A column 24 h later. Two copies of the DDD2-mEGFR are attachedto each AD2 moiety, resulting in a DNL complex comprising one hLL1 IgGmoiety and four mEGFR xenoantigen moieties.

Administration of DNL vaccine anti-CD74-mEGFR to subjects withEGFR-expressing NSCLC induces an immune response against EGFR-expressingcancer stem cells. The immune response is effective to reduce oreliminate EGFR positive cancer cells in the subjects.

The skilled artisan will realize that DNL-based vaccines incorporatingxenoantigen moieties corresponding to a wide variety of tumor-associatedantigens may be constructed and utilized according to the techniquesdescribed herein.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and used without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the METHODS described hereinwithout departing from the concept, spirit and scope of the invention.More specifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

What is claimed is:
 1. A complex comprising: a) an antibody moiety thatbinds to a dendritic cell antigen selected from the group consisting ofCD209 (DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like receptor 2), TLR 4,TLR 7, TLR 9, BDCA-2, BDCA-3, BDCA-4, and HLA-DR, wherein the antibodymoiety is attached to a DDD (dimerization and docking domain) moietyfrom human protein kinase A RIα, RIβ, RIIα or RIIβ; and b) a xenoantigenmoiety attached to an AD (anchor domain) moiety from an AKAP (A-kinaseanchoring protein); wherein two copies of the DDD moiety form a dimerthat binds to the AD moiety to form the complex.
 2. The complex of claim1, wherein the antibody moiety is an anti-CD74 antibody orantigen-binding fragment thereof.
 3. The complex of claim 1, wherein thexenoantigen is selected from the group consisting of carbonic anhydraseIX, alpha-fetoprotein, α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55,CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126,CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, colon-specificantigen-p (CSAp), CEA (CEACAM5), CEACAM6, DAM, EGFR, EGFRvIII, EGP-1,EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen,GAGE, gp100, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG)and its subunits, HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1),HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R,IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18,IL-25, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE,MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B,MIF, MUC1, MUC2, MUC3, MUC4, MUM-1/2, MUM-3, NCA66, NCA95, NCA90,antigen specific for PAM-4 antibody, placental growth factor, p53,prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6,IL-25, RS5, RANTES, T101, SAGE, S 100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, cMET, an oncogene markerand an oncogene product.
 4. The complex of claim 1, wherein thexenoantigen is CD20.
 5. The complex of claim 1, wherein the amino acidsequence of the DDD moiety is selected from the group consisting of SEQID NO:1 and SEQ ID NO:2.
 6. The complex of claim 1, wherein the aminoacid sequence of the AD moiety is selected from the group consisting ofSEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ IDNO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ IDNO:15 and SEQ ID NO:16.
 7. The complex of claim 1, wherein the antibodymoiety is selected from the group consisting of an IgG antibody and anantigen-binding antibody fragment.
 8. The complex of claim 1, whereinthe antibody moiety is a humanized or chimeric anti-CD74 antibody orantigen-binding fragment thereof comprising the light chain variablecomplementarity-determining region (CDR) sequences CDR1(RSSQSLVHRNGNTYLH; SEQ ID NO:1), CDR2 (TVSNRFS; SEQ ID NO:2), and CDR3(SQSSHVPPT; SEQ ID NO:3) and the heavy chain variable region CDRsequences CDR1 (NYGVN; SEQ ID NO:4), CDR2 (WINPNTGEPTFDDDFKG; SEQ IDNO:5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6)
 9. The complex of claim 4,wherein the amino acid sequence of the CD20 xenoantigen moiety is SEQ IDNO:7.
 10. The complex of claim 1, wherein the antibody moiety attachedto a DDD moiety forms a first fusion protein and the xenoantigen moietyattached to an AD moiety forms a second fusion protein.
 11. The complexof claim 1, wherein the complex is capable of inducing an immuneresponse against CD138^(neg)CD20⁺ MM stem cells when administered to asubject.
 12. A complex comprising: c) an antibody moiety that binds to adendritic cell antigen selected from the group consisting of CD209(DC-SIGN), CD34, CD74, CD205, TLR 2 (toll-like receptor 2), TLR 4, TLR7, TLR 9, BDCA-2, BDCA-3, BDCA-4, and HLA-DR, wherein the antibodymoiety is attached to an AD moiety, wherein the AD (anchor domain)moiety from an AKAP (A-kinase anchoring protein); and d) a xenoantigenmoiety attached to a DDD moiety, wherein said DDD (dimerization anddocking domain) moiety from human protein kinase A RIα, RIβ, RIIα orRIIβ; wherein two copies of the DDD moiety form a dimer that binds tothe AD moiety to form the complex.
 13. The complex of claim 12, whereinthe antibody moiety is an anti-CD74 antibody or antigen-binding fragmentthereof.
 14. The complex of claim 12, wherein the xenoantigen isselected from the group consisting of carbonic anhydrase IX,alpha-fetoprotein, α-actinin-4, A3, antigen specific for A33 antibody,ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m,CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15,CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33,CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64,CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138,CD147, CD154, CDC27, CDK-4/m, CDKN2A, colon-specific antigen-p (CSAp),CEA (CEACAM5), CEACAM6, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M,Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GROB,HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits,HER2/neu, HMGB-1, hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, Ia,IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R,IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, insulin growthfactor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, LDR/FUT,macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1,MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2,MUC3, MUC4, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, antigen specific forPAM-4 antibody, placental growth factor, p53, prostatic acidphosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6, IL-25, RS5,RANTES, T101, SAGE, S100, survivin, survivin-2B, TAC, TAG-72, tenascin,TRAIL receptors, TNF-α, Tn antigen, Thomson-Friedenreich antigens, tumornecrosis antigens, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen,complement factors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2,bcl-6, Kras, cMET, an oncogene marker and an oncogene product.
 15. Thecomplex of claim 12, wherein the xenoantigen is CD20.
 16. The complex ofclaim 12, wherein the amino acid sequence of the DDD moiety is selectedfrom the group consisting of SEQ ID NO:1 and SEQ ID NO:2.
 17. Thecomplex of claim 12, wherein the amino acid sequence of the AD moiety isselected from the group consisting of SEQ ID NO:3; SEQ ID NO:4; SEQ IDNO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ IDNO:12; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15 and SEQ ID NO:16. 18.The complex of claim 12, wherein the antibody moiety is selected fromthe group consisting of an IgG antibody and an antigen-binding antibodyfragment.
 19. The complex of claim 12, wherein the antibody moiety is ahumanized or chimeric anti-CD74 antibody or antigen-binding fragmentthereof comprising the light chain variable complementarity-determiningregion (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:1), CDR2(TVSNRFS; SEQ ID NO:2), and CDR3 (SQSSHVPPT; SEQ ID NO:3) and the heavychain variable region CDR sequences CDR1 (NYGVN; SEQ ID NO:4), CDR2(WINPNTGEPTFDDDFKG; SEQ ID NO:5), and CDR3 (SRGKNEAWFAY; SEQ ID NO:6)20. The complex of claim 15, wherein the amino acid sequence of the CD20xenoantigen moiety is SEQ NO:7.
 21. The complex of claim 12, wherein theantibody moiety attached to a DDD moiety forms a first fusion proteinand the xenoantigen moiety attached to an AD moiety forms a secondfusion protein.
 22. The complex of claim 12, wherein the complex iscapable of inducing an immune response against CD138^(neg)CD20⁺ MM stemcells when administered to a subject.